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Purification and characterization of the human hemopoietic stem cell Sutherland, Heather Jeanine 1991

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PURIFICATION AND CHARACTERIZATION OF T H E H U M A N HEMOPOIETIC S T E M C E L L by H E A T H E R JEANINE SUTHERLAND BSc-Honors, University of Saskatchewan, 1975 M . D . , University of Saskatchewan, 1980 A THESIS SUBMITTED IN PARTIAL F U L F U L L M E N T O F T H E REQUIREMENTS F O R T H E D E G R E E O F D O C T O R OF PHILOSOPHY In T H E FACULTY OF GRADUATE STUDIES (Department of Pathology) We accept this thesis as conforming to the required standard T H E UNIVERSITY OF BRITISH COLUMBIA February, 1991 © Heather Jeanine Sutherland, 1991 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department The University of British Columbia Vancouver, Canada Date DE-6 (2/88) 11 ABSTRACT Previous studies In mice have suggested that some if not all hemopoietic stem cells with long-term in vivo repopulating ability are biologically, physically, and pharmacologically different from cells detectable by short-term colony assays. Since human hemopoietic stem cells cannot be assessed by expression of their in vivo repopulating potential, characterization of these cells requires an alternative endpoint. This thesis explores the use of clonogenic cell production in vitro in the presence of a competent stromal cell feeder layer for this purpose, based on the observation that this can continue for many weeks when unseparated human marrow cells are cultured under conditions that allow a stromal cell layer to form. Accordingly, a population of human clonogenic cell precursors referred to as long-term culture-initiating cells (LTC-IC) were postulated to exist as a biologically distinct compartment whose members could be quantitated by measuring the number of myeloid, erythroid and multi-lineage clonogenic progenitors present after 5 weeks of their culture on stromal feeder layers. LTC-IC in normal marrow assayed in this way were found to have a significantly lower forward light scatter, lower expression of HLA-DR, lower expression of CD 71 (transferrin receptor), and a higher expression of CD 34 as compared to clonogenic cells. Separation of marrow cells on the basis of these differences allowed a cell population enriched -800 fold in LTC-IC to be obtained. This population contained only 0.06% of the marrow cells and 2% of the total clonogenic cells, but retained 50 - 60% of the LTC-IC present in the original marrow. Absolute numbers of LTC-IC and the proliferative and differentiative capability of individual LTC-IC were then determined by limiting dilution analysis following the demonstration that clonogenic cell output (at 5 weeks) is linearly related to input cell number over a wide range of cell concentrations. The frequency of LTC-IC in normal human marrow was determined to be ~1 per 2 x 10^ cells. Following purification this was increased to 1-2%. The proliferative capacity exhibited by individual LTC-IC as measured by the number of ill clonogenic cells per LTC-IC in 5 week-old cultures was found to range from 1 to 30 (the average being ~4). These studies also showed that a least some LTC-IC are multipotent as evident by their production of both erythroid and myeloid progeny. To study the effect of specific growth factors on LTC-IC maintenance and differentiation, highly purified LTC-IC were seeded onto irradiated murine marrow-derived stromal cells (from the M2-10B4 line) previously engineered to produce one of the human hemopoietic growth factors G-CSF, GM-CSF or IL-3. In the absence of any feeders, both the LTC-IC and their progeny in these purified suspensions decreased to very low levels within 5 weeks. However, in the presence of control M2-10B4 cells, LTC-IC maintenance and differentiation was supported as effectively as when standard human marrow feeders were present. The combined presence of G-CSF and IL-3-producing M2-10B4 cells further enhanced the maintenance and early differentiation of LTC-IC above levels obtained with control feeders, but only in the absence of GM-CSF producing feeders. In contrast, in the presence of GM-CSF-producing feeders the output of mature granulocytes and macrophages was maximal, and LTC-IC were inhibited. These studies describe and validate the use of the LTC-IC assay to selectively identify and quantitate a previously inaccessible population of very primitive human hemopoietic cells. Exploration of in vitro conditions and human growth factors able to influence their developmental behaviour points to G-CSF plus 11-3 as the best candidates for future studies of LTC-IC activation and/or expansion in vitro. iv TABLE OF CONTENTS Page ABSTRACT ii TABLE OF CONTENTS iv LIST OF TABLES vii LIST OF FIGURES viii LIST OF ABBREVIATIONS ix ACKNOWLEDGMENTS x CHAPTER I INTRODUCTION 1 1. Organization of the hemopoietic system 1 A. Development and structure of the bone marrow 1 B. Hierarchical differentiation pattern of hemopoiesis 3 2. Quantitation of hemopoietic progenitor cells 7 A. In vivo repopulation assays for murine stem cells 7 B. Clonogenic assays 9 C. Long-term bone marrow culture 13 3. Purification of primitive human hemopoietic cells * 18 A. Physical separation techniques 18 B. Light scatter properties of primitive cells 20 C. Use of anti-CD 34 monoclonal antibodies 23 D. Other monoclonal antibodies used for the purification of primitive hemopoietic cells 24 4. Growth factor regulation of hemopoiesis 26 A. Colony-stimulating factors 26 B. Interleukins 30 5. Stromal cell regulation of hemopoiesis 32 6. Thesis objectives 36 References 38 CHAPTER II MATERIALS AND METHODS 54 1. Cells 54 A. Bone marrow cells 54 B. Cell line maintenance 55 2. Staining and flow cytometry 56 A. Studies of light scatter, HLA-DR and CD 34 reactivity of clonogenic cells and LTC-IC (Chapters III, V, VI) 56 B. Studies using Workshop antibodies (Chapter IV) 57 3. Clonogenic assays 58 4. Long-term marrow cultures 58 5. Limiting dilution analysis using long-term cultures 59 6. Retrovirally-infected M2-10B4 cells 60 7. Co-cultures using engineered M2-10B4 cells 61 8. Growth factor bioactivity assays 62 References 63 V CHAPTER III CHARACTERIZATION AND PARTIAL PURIFICATION OF HUMAN MARROW CELLS CAPABLE OF INITIATING LONG-TERM HEMOPOIESIS IN VITRO 65 1. Introduction 65 2. Results 66 A. Density separation 66 B. Light scatter properties of clonogenic cells and LTC-IC 67 C. HLA-DR expression on clonogenic cells and LTC-IC 70 D. Progenitor enrichment by sorting for high CD 34 expression 72 E. Partial purification of clonogenic cells and LTC-IC 73 3. Discussion 77 References 81 CHAPTER IV DIFFERENTIAL EXPRESSION OF ANTIGENS ON CELLS THAT INITIATE HEMOPOIESIS IN LONG-TERM HUMAN MARROW CULTURE 83 1. Introduction 83 2. Results 83 3. Discussion 87 References 88 CHAPTER V FUNCTIONAL CHARACTERIZATION OF INDIVIDUAL HUMAN HEMOPOIETIC STEM CELLS CULTURED AT LIMITING DILUTION ON SUPPORTIVE MARROW STROMA 89 1. Introduction 89 2. Results 90 A. Stromal feeder requirement of human LTC-IC 90 B. Clonogenic progenitor output is linearly related to the number of marrow cells assayed 91 C. Quantitation of LTC-IC by limiting dilution analysis 94 D. Proliferative properties of LTC-IC 97 E. Differentiative properties of LTC-IC 101 3. Discussion 102 References 105 vi CHAPTER VI DIFFERENTIAL REGULATION OF SEQUENTIAL STAGES OF HUMAN HEMOPOIESIS IN LONG-TERM CULTURES OF H I G H L Y PURIFIED H E M O P O I E T I C S T E M C E L L S MAINTAINED ON GENETICALLY ENGINEERED MURINE STROMAL CELLS 107 1. Introduction 107 2. Results 108 A. Growth factor production by engineered M2-10B4 cells 108 B. Capacity of M2-10B4 cells to support human hemopoiesis 110 C. Specific growth factor effects on terminal hemopoiesis 111 D. Specific growth factor effects on clonogenic cell output 113 E. Specific growth factor effects on LTC-IC maintenance 115 F. Lack of any growth factor effect on the proliferative potential displayed by LTC-IC present after 5 weeks in culture 117 3. Discussion 118 References 123 CHAPTER VII SUMMARY AND FUTURE DIRECTIONS 126 References 135 vii LIST OF TABLES Frequency of Clonogenic Cells and LTC-IC in Human Marrow Before and After Density Centrifugation on 1.066 - 1.068 gm/cm^ Percoll. Degree of Enrichment of Clonogenic and LTC-IC After Sorting of Low Density Human Marrow for High My 10 Expression. Selective Enrichment of Clonogenic Cells in Normal Human Marrow by Multi-parameter Sorting. Selective Enrichment of LTC-IC in Normal Human Marrow by Multi-parameter Sorting. Relative Proportions of Different Types of Clonogenic Cells Detected Before and After 5 Weeks in LTC (% of Total). Percentage of Clonogenic and LTC-IC in Fractions Sorted According to Staining with Workshop Monoclonal Antibodies. Linearity of Clonogenic Progenitor Numbers After 5 Weeks in LTC as a Function of the Number of Cells Seeded per LTC. Absolute Frequencies of LTC-IC. Proliferative Potential of LTC-IC. Growth Factor Production by Retrovirally-Infected M2-10B4 Cells. Content of 5 Week Co-Cultures on M2-10B4 Feeders as Compared to Human MF and No Feeders (Plastic). Number of Clonogenic Progenitors Per LTC-IC Harvested From 5 Week-Old Co-cultures Containing Different Types of Feeder. v l i l LIST OF FIGURES page Figure 1. Assay for LTC-IC. 17 Figure 2. 2A: Light scatter properties of normal human peripheral blood. 2B: Light scatter properties of normal human bone marrow. 22 Figure 3. Light scatter properties of clonogenic cells and LTC-IC. 69 Figure 4. Expression of HLA-DR on clonogenic cells and LTC-IC. 71 Figure 5. Enrichment of clonogenic and LTC-IC as compared to whole bone marrow by density centrifugation and FACS sorting. 78 Figure 6. Fluorescence profiles of light-scatter gated low density human bone marrow cells stained indirectly with Workshop monoclonal antibodies. 86 Figure 7. Model of hemopoiesis in LTC. 90 Figure 8. Input-output relationship in LTC. 93 Figure 9. Limiting dilution analysis. 96 Figure 10. Number of clonogenic progenitors produced by individual LTC-IC. 100 Figure 11. TyP e of clonogenic progenitors produced by individual LTC-IC. 101 Figure 12. The number of nonadherent (NA) cells in 5 week-old co-cultures. 112 Figure 13. The number of clonogenic cells in 5 week-old co-cultures. 114 Figure 14. The number of LTC-IC's as determined by limiting dilution analysis in 5 week-old co-cultures. 116 Figure 15. Influence of growth factor producing feeders on three levels of hemopoiesis occuring over 5 weeks in LTC. 119 lx LIST OF ABBREVIATIONS BFU-E burst-forming-unit-erythroid CD cluster determinants CFU-E colony-formlng-unit-erythroid CFU-G colony-forrning-unit-granulocyte CFU-GEMM colony-formlng-unlt-granulocyte / erythrold/macrophage /megakaryocyte CFU-GM colony-fomilng-unit-granulocyte/macrophage CFU-M colony-formlng-unit-macrophage CFU-Meg colony-formlng-unlt-megakaryocyte (CFU-Mk) CFU-S colony-fonxilng-unlt-spleen CML chronic myelogenous leukemia cm centimeters CSF-1 colony-stimulating-factor-1 (M-CSF) DNA deoxyribonucleic acid DMEM Dulbecco's modified Eagle's medium FACS fluorescence activated cell sorter FCS fetal calf serum FITC fluorescein isothiocyanate FLS forward light scatter g grams G418 r G418 resistant G-CSF granulocyte-colony-stimulating-factor GM-CSF granulocyte /macrophage -colony-stimulating-factor Gy Gray 4-HC 4-hydroperoxycyclophosphamlde HFN Hank's buffered saline with 2% fetal calf serum and 0.1% sodium azide HLA-DR human leukocyte antigen-DR IL- interleukin kb kilobase LTC long-term culture LPS lipopolysaccharide LTC-IC long-term culture-initiating cell MF marrow feeders ml milliliter mRNA messenger ribonucleic acid NA nonadherent nm nanometers OLS orthogonal light scatter PDGF platelet-derived-growth-factor PE phycoerythrin PI propridium iodide RNA ribonucleic acid SAM sheep anti-mouse IgG SEM standard error of the mean Tdt terminal deoxynucleotidyl transferase TGF-P transforming growth factor-beta tk thymidine kinase U units X ACKNOWLEDGEMENTS I would like to express my sincere gratitude to my research supervisor Dr. Connie Eaves and my co-supervisor Dr. Peter Lansdorp for their guidance, encouragement, support and patience during this project. I would also like to thank Dr. Allen Eaves and Dr. Donna Hogge for helpful discussions and collaborations. I also wish to thank Sara Abraham, Coleen McAloney, Visia Dragowska and Darlene Heath, Karen Lambie and Birgitte Gerhard for excellent technical assistance and Don Henkelman for statistical advice. This work was supported by grants from the Medical Research Council of Canada and the National Cancer Institute of Canada (NCIC) with core support from the B.C. Cancer Foundation and the B.C. Cancer Agency. I was a Terry Fox Physician-Scientist Training Fellow of the NCIC and gratefully acknowledge their financial support. 1 C H A P T E R I INTRODUCTION 1. ORGANIZATION O F T H E HEMOPOIETIC SYSTEM (A) Development and Structure of the Bone Marrow The bone marrow, beginning in the third trimester of gestation, is the principal site of blood cell formation in humans. In the normal adult it is responsible for the daily production of about 2.5 billion red cells, 2.5 billion platelets and 1.0 billion neutrophils per kilogram body weight. These are necessary to replace senescent cells removed daily from the circulation. 1 This life-long process of blood cell production is due to the regulated activity of hemopoietic stem cells that can self-renew as well as produce progenitors capable of differentiating along all of the blood cell lineages. The mechanisms regulating stem cell behavior are the subject of intense investigation as they have implications for many hematological diseases, and if understood would provide insight as to the molecular basis of differentiation versus self-renewal decisions. Hemopoiesis begins very early in development, with evidence for erythropoiesis in the human embryo at 19 days gestation. These erythroid cells are thought to differentiate from the earliest hemopoietic cells, referred to as hemocytoblasts, which are of mesodermal origin arising from the vessels of the yolk sac.^ Subsequently, certain primitive yolk sac cells migrate to the embryonic liver^ which then becomes the major source of red blood cells until the bone marrow takes over this f u n c t i o n at about 24 weeks gestation. G r a n u l o p o i e s i s a n d 2 megakaryopoiesis occur transiently in the fetal liver, but exist predominantly in the bone marrow from the 12th week of gestation onward. At birth the bone marrow is essentially the only site of hemopoiesis.1 Gradually during childhood the long bones are filled with fat cells and hemopoiesis becomes limited to the vertebrae, pelvis, ribs and skull during adult life. Lymphocytes originate from stem cells 4 which can be detected in hemopoietic tissue in the yolk sac, fetal liver and spleen and subsequently bone marrow. T lymphocyte precursors migrate to the thymus where they complete their differentation. B lymphocytes are thought to mature in the fetal liver beginning at 8 to 9 weeks of gestation in man, and subsequently in the adult bone marrow. 5 , 6 T lymphopoiesis in the thymus begins with a pre T-cell which is CD 4 and CD 8 negative (CD 8-) and contains terminal deoxynucleotidyl transferase (Tdt). These cells subsequently differentiate under the control of the thymic microenvironment and growth factors to generate functionally mature CD 4+ CD 8- T helper cells and CD 4- CD8+ T suppressor/cytotoxic cells. Mature B cells migrate to the peripheral lymphoid tissues which include the lymph nodes, spleen, and mucosal-associated lymphoid tissues. There, under the influence of antigen and T helper cells, they are stimulated to differentiate into immunoglobulin-producing plasma cells. 6 That part of the bone marrow where hemopoiesis occurs, or "red" marrow, consists of hemopoietic cells lying in a meshwork of venous sinuses and branched fibroblastic cells. The wall of the sinuses is composed of a luminal layer of endothelial cells and an incomplete layer of reticular cells. 7 The reticular cells synthesise reticular fibers which, together with their cytoplasmic processes, form a physical structure within which hemopoiesis occurs. Bone marrow fibroblasts and adipocytes are thought to be derived from reticular cells and are of nonhemopoietic origin. 1- 7"^ Upon stimulation with stromal cell activators such as IL-1 these mesenchymal cells can be induced to produce hemopoietic growth factors which are thought to be important in the control of local hemopoiesis. 1 0" 1 3 Marrow stroma cells secrete a complex 3 extracellular matrix that is composed of both fibrous and non fibrous proteins including collagen types I, III and IV, fibronectin, and l a m i n i n . 1 4 , 1 5 Glycosaminoglycans are also present in extracellular matrix and one component, heparan sulfate but not chondroitin sulfate, can bind the hemopoietic growth factors G M - C S F and IL-3 suggesting the potential role of extracellular matrix in the presentation of growth factors to hemopoietic c e l l s . 1 6 In most t issues glycosaminoglycans is complexed to core proteins to form p r o t e o g l y c a n s . 1 5 Erythropoiesis occurs in erythropoietic islands with a central histiocyte (macrophage) which phagocytoses the extruded nuclei and may have a role in the provision of nutrients or growth factors to the erythrocyte p r e c u r s o r s . 7 ' 1 7 Megakaryocytes tend to be clustered close to the venous s inuses . 7 Mature hemopoietic cells enter the circulation via small holes through the cytoplasm of endothelial cells under the control of as yet poorly characterized releasing factors which may include complement, glucocorticoid or androgenic hormones, or endotoxin. 1 ^ The overall production and egress of mature hemopoietic cells from the bone marrow into the circulation must be tightly controlled, possibly by a combination of glycoprotein growth factors, stromal factors or other mechanisms that couple hemopoiesis to the needs of the body. (B) Hierarchical Differentiation Pattern of Hemopoiesis Mature myeloid cell production involves a lengthy differentiation process spanning many cell generations during which the progeny of a single hemopoietic stem cell, located in the bone marrow, may be greatly amplified. 19,20 Although the differentiation process is viewed as a continuous process, the cells which are the precursors of mature blood cells are often divided into three functional subdivisions. Myeloid cells which can be morphologically recognized as belonging to specific lineages and comprise the last 3 to 5 divisions of development along those lineages are termed precursor cells. These cells are committed to complete their differentiation during a relatively fixed number of cell cycles and have no self-renewal capability. Cells 4 representing earlier stages of differentiation are rare in the bone marrow (1 per 10 3 to 1 per 10 4 cells) and cannot be uniquely recognized by their morphology. However, they can be identified by functional endpoints which measure the number and types of progeny they generate. One such functional assay, which Is used to define the second level of hemopoietic differentiation, is the in vitro clonogenic assay. It is used to identify cells capable of dividing in semi-solid media to produce a clone which can then be recognized.^1_^3 Most such clonogenic progenitors are restricted in their differentiation potential to one or a subset of all the blood cell lineages and have an intermediate but finite proliferative capacity (up to 15 to 20 divisions) which varies with the progenitor subtype. The earliest stage level of hemopoiesis is the stem cell compartment. These cells have very extensive proliferative potential such that they can generate progeny that will reconstitute hemopoiesis in an irradiated host, and have self-renewal and toti-potential differentiative capability.^ 4 Within each of these levels of hemopoiesis, further hierarchical subdivisions can also be defined. In the assessment of precursor cells using bone marrow morphology, the earliest identifiable erythroid precursor is the pronormoblast.17 This cell subsequently undergoes about 4 cell divisions to produce first basophilic normoblasts, and then an average of 16 (range 8 to 32) polychromatophilic normoblasts. Subsequent maturation involves no further cell division. The first recognizable precursor of mature granulocytes (neutrophils, eosinophils and basophils) is the myeloblast, which also is thought to undergo about 4 further divisions to produce sequentially: promyelocytes, myelocytes, and then metamyelocytes. After this stage further cytoplasmic and nuclear maturation results in the formation of functional granulocytes. Terminal differentiation of megakaryocytes usually involves polyploidization such that platelets are produced by cytoplasmic budding from a 16N or 32N mature megakaryocyte. 5 Over the past 25 years, culture conditions have been developed employing semi-solid media that support the formation in a single culture of multiple colonies of mature red cells, granulocytes, macrophages and megakaryocytes, or combinations of these, each from isolated single cells. These colony assays have thus provided the ability to analyse both quantitatively and qualitatively a variety of progenitor cell types present in both the peripheral blood and bone marrow of normal adults. Serum (or the essential constituents thereof) and a source of growth factors are usually provided to allow expression of the maximum proliferative potential of the particular progenitors to be detected. Analysis of human hemopoiesis using such colony assay systems has helped to establish two important principles first evolved from studies of murine cells. First, the majority of nucleated cells in any normal hemopoietic tissue are in the terminal stages of morphological maturation and no longer have sufficient proliferative p o t e n t i a l to give r ise to a s c o r a b l e c o l o n y (ie. a c o l o n y c o n t a i n i n g m o r e t h a n 2 m e g a k a r y o c y t e s , 2 5 , 2 6 8 erythroblasts , 2 7 or 20-50 granulocytes a n d / o r macrophages 2 8 ) . Second, the considerable heterogeneity i n both the size and the composition of colonies observed when unfractionated marrow samples are assessed even under optimal plating conditions for a particular lineage is due to a corresponding heterogeneity in the populations of progenitors stimulated. The greater the proliferative and differentiative potential displayed, the more primitive is the progenitor type represented. 2^ Early studies i n mice indicated the existence i n adult mouse marrow of pluripotent hemopoietic cells able to produce colonies in the spleens of transplanted recipients which contained progeny capable of differentiating along multiple myeloid lineages.30.3 1 These spleen colony-forming cells were shown to generate new pluripotent spleen colony-forming cells indicating some self-renewal capacity.^ 1 Later using chromosomal markers these cells were shown to derive from a cell in adult marrow that also gives rise to lymphoid cel ls . 4 With the introduction of retroviral marking strategies to allow the more extensive identification in vivo of the progeny of individual initial cells, it was shown that single cells can regenerate both 6 lymphoid and myeloid tissues following transplantation in vivo and can sustain the production of all blood cell types for extensive periods of t i m e . 3 ^ " 3 4 Comparison of the properties of m u r i n e hemopoietic cells detected by i n vitro or i n vivo clonogenic assays a n d their relationships to transplantable long-term reconstituting pluripotent hemopoietic cells have established some overlap between all three, with the latter representing the most primitive p o p u l a t i o n . 3 5 " 3 ^ Evidence of such hierarchy has been most recently demonstrated as a difference in the abilities of separable populations to achieve rapid as opposed to long-term hemopoietic reconstitution after transplantation. 4 0 Even amongst cells that can sustain long-term hemopoiesis in vivo a hierarchy can be revealed if the cells are tested in a competitive repopulation a s s a y . 4 1 " 4 3 Initial evidence for the existence of hemopoietic cells i n m a n with lymphoid and myelopoietic potential was derived from analyses of the expression of the X-linked isoenzyme glucose-6-phosphate dehydrogenase (G6PD) in heterozygous women with myelodysplastic or myeloproliferative diseases. Clonal involvement of both the myeloid and lymphoid lineages found in some cases suggested that malignant transformation had occurred in a cell with both p o t e n t i a l i t i e s . 4 4 " 4 6 The presence of marker chromosomes (such as the Phi ladelphia chromosome) in isolated lymphoid and myeloid cell subpopulations from the same patient has also supported this interpretation. 4 7 More recently, monoclonal donor-derived regeneration of lymphoid and myeloid cell populations in recipients of allogeneic marrow transplants has been d e m o n s t r a t e d 4 ^ i n d i c a t i n g the presence of a cell with both l y m p h o i d a n d m y e l o i d reconst i tut ing capabili t ies i n n o r m a l adult h u m a n bone marrow. A s s a y s that allow quantitation of totipotent cells in the mouse are beginning to be introduced and v a l i d a t e d , 4 3 and the development of assays for analogous h u m a n cells is currently a n area of active investigation. 7 2. QUANTITATION O F HEMOPOIETIC PROGENITOR CELLS (A) In Vivo Repopulation Assays for Murine Stem Cells In 1961 Till and McCulloch described an in vivo colony assay that allowed the detection of murine pluripotent hemopoietic cells.3*-* In this assay femoral marrow cells from normal mice were injected into syngeneic mice after they recieved 950 rads of y-irradiation. After 8 to 14 days in recipients that recieved 1 0 4 to 10 5 bone marrow cells, nodules were visible on the surface of the spleen. Morphological examination of the cells within these nodules revealed the presence of granulopoiesis, erythropoiesis and megakaryopoiesis within a single n o d u l e . 4 ^ Using donor cells which had unique radiation-induced chromosomal markers it was shown that these nodules were derived from a single cell, as all metaphases within an individual spleen colony had the same chromosomal m a r k e r . 5 ^ The cell which gave rise to this colony could thus be assumed to be pluripotent with a proliferative capacity sufficient to give rise to a colony of ~ 1 0 6 cells. Moreover, when individual spleen nodules were injected into secondary recipients, in some cases new multi-lineage spleen nodules developed demonstrating self-renewal of the original colony-forming c e l l . 3 1 The term "colony forming unit-spleen" (CFU-S) was introduced for the cells which were responsible for producing these nodules. Because only a fraction of the cells in any injected cell preparation lodge in the spleen, quantitation of spleen nodules will underestimate the true total number of cells with spleen colony-forming potential. To obtain an estimate of the proportion of cells with spleen colony forming potential that actually lodge in the spleen and thus provide a way of calculating the original number of cells with such potential in a given starting population, the number of spleen nodules produced in secondary recipients after injection of cells removed from the spleens of primary recipients soon after their injection (upon completion of seeding but prior to growth) may be divided by the number of spleen nodules produced in primary recipients. Values ranging from 3-19% have been reported by various investigators depending upon the details of the protocol u s e d . 3 1 , 5 1 It 8 has also been shown that C F U - S i n steady state murine bone marrow are a quiescent population as shown by their resistance to killing by a short exposure to high specific activity thymidine or other S-phase specific cytotoxic a g e n t s . 3 0 More recent studies with long-term exposure to BUdr indicate C F U - S to be a slowly turning over populat ion. 5 ^ i n regenerating bone marrow (as in the post-bone marrow transplant situation) the proportion of C F U - S in S-phase is markedly increased. 3 0 Subsequently, a hierarchy was identified within the normal marrow C F U - S population. This was first suggested by cell separation studies in which self-renewal capacity was shown to increase with decreasing cell s i z e . 5 3 Later spleen nodules detected 7 to 9 days after injection of normal marrow were found to contain cells of a single lineage and no C F U - S . 5 4 Cell populations enriched for day 7 C F U - S have also been found to have a low radioprotective a b i l i t y . 5 5 In contrast, spleen colonies appearing at later times were mixed and did contain C F U - S . Thus most efforts to purify and characterize murine stem cells then focussed on the day 12 to 14 C F U - S assay on the assumption that this detected the most primitive cell typ e 37-39 However, more recently evidence of further hierarchy has emerged. For example, a cell distinct from C F U - S but which gives rise to C F U - S has been identified. This cell (termed pre-CFU-S) is assayed by its ability to produce a high number of C F U - S in the bone marrow of recipients 13 days after injection into lethally irradiated h o s t s . 5 6 , 5 7 Retroviral marking of murine bone marrow transplants has allowed the initial dynamic behavior of individual stem cell clones to be observed.24,32,58 Such studies have also now shown that an individual stem cell can eventually repopulate and maintain both lymphoid and myeloid tissues for very long periods of time, although during the first several months, the sequential activation of different clones with only a few (1 or 2) clones contributing to hemopoiesis at each point in time may be observed.^ 4 Qualitative differences may exist between the cells that contribute to early as opposed to late reconstitution of hemopoiesis . 5 9 9 The latter co-purify better with p r e - C F U - S 6 0 and the former with C F U - S . It has also been f o u n d that cel ls that have previously undergone two cycles of ser ia l bone marrow transplantation and regeneration, although capable of regenerating and sustaining normal hemopoiesis when injected alone, are consistently out-competed by marrow cells from previously untreated donors when mixed grafts are g i v e n . 4 3 , 5 ® Thus factors other than time must also be considered i n attempts to define hierarchy within the hemopoietic stem cell compartment. B. Clonogenic assays In in vitro clonogenic assays, colony formation depends on the provision of appropriate nutrients and growth factor support for expression of the proliferative and differentiative potential of the progenitors to be detected. A s these vary for different types of progenitors, conditions appropriate for one may or may not be appropriate for, or even compatible with another. Conditions that support the in vitro clonal growth and maturation of most types of h u m a n myeloid p r o g e n i t o r s 2 1 " 2 3 and mature h u m a n B 6 1 and T c e l l s 6 2 have now been recognized. In contrast, conditions that allow early stages of lymphopoiesis to occur in a clonal assay system are not well defined even in the murine system, although recently some progress has been made for the B - l i n e a g e . 6 3 - 6 5 For clonogenic cell assays to be useful it must be possible to plate cells at a sufficiently low density to allow discrete colonies to be individually evaluated and scored. For such assays to be quantitative a linear relationship between the number of cells plated and the number of colonies produced must be achieved. Erythropoietic progenitors generate colonies that at the end of their growth phase are made up of clusters of hemoglobinized erythroblasts. The number of such clusters allows erythropoietic progenitors to be subdivided into different classes of different maturity. The 10 more mature are cells referred to as colony-foniilng-unlts-erythroid (CFU-E), the more primitive as burst-forming-units-erythroid (BFU-E). H u m a n C F U - E have been defined as cells that produce only one or two clusters of cells, each consisting of from 8 to -100 hemoglobinized erythroblasts. Maturation of these cells is complete on average 10 to 12 days after initiation of the culture. Thereafter, these small erythroid colonies become more difficult to identify as the cells within them begin to lyse and CFU-E-derived colony counts thus appear to d e c l i n e . 2 7 , 6 6 B F U - E may be readily subdivided into those that ultimately produce colonies that are relatively small (containing 3 to 8 clusters of erythroblasts)(mature BFU-E), of intermediate size (9 to 16 clusters)(intermediate B F U - E ) , or large (greater than 16 clusters)(primitive B F U - E ) . Small bursts, on average, mature earlier and are best counted at the same time as colonies derived from C F U - E ; whereas the intermediate and large bursts mature later, and are optimally evaluated 18 to 20 days after culture initiation. The division between small, intermediate and large bursts also has a physiological basis. Mature B F U - E , like C F U - E , represent more rapidly turning over populations in normal marrow than intermediate and primitive B F U - E and are physically larger c e l l s . 2 ^ 6 6 , 6 7 Clonogenic progenitors of granulocytes (CFU-G), macrophages (CFU-M) or both (CFU-GM) are defined on the basis of their ability to produce colonies containing a minimum of 20 (or 50) mature cells. For granulocytic progenitors, evidence of a hierarchy of cells of decreasing proliferative potential has also been o b t a i n e d . 6 8 , 6 ^ However, due to the more homogeneous star-like morphology of the colonies generated by all classes of granulopoietic progenitors and the greater dependence of colony size and composition on culture conditions (by comparison to erythroid colony formation), it is not as easy to develop absolute colony scoring criteria that allow reliable progenitor subtype assignment. Nevertheless, when h u m a n leukocyte conditioned medium is used as a standardized source of granulocyte colony-stimulating factors, separate scoring of very large colonies (containing more than 500 cells after 2 1/2 weeks of incubation) has allowed the recognition of a clonogenic granulopoietic progenitor that. 11 like primitive B F U - E , are quiescent in normal human bone marrow in contrast to the majority of clonogenic granulocytic progenitors that generate smaller colonies under these conditions and represent a population in normal bone marrow that is continuously proliferating. 7 0 Pure megakaryocyte colonies (CFU-Meg or CFU-Mk) resemble macrophages in unstained preparations and are thus best identified by staining with lineage specific markers. Since terminal amplification of megakaryocytes involves polyploidization, a clonogenic cell that produced two megakaryocytes may be viewed as analogous to a clonogenic erythroid or granulopoietic cell capable of undergoing 3 to 5 divisions. A s for the other lineages large as well as small megakaryocyte colonies have been defined and assumed to derive from primitive and more mature megakaryocyte progenitors, r e s p e c t i v e l y . 7 1 , 7 2 Colonies containing multiple lineages of mature cells can also be generated under conditions optimized for the generation of pure erythroid, granulopoietic and megakaryocytic colonies. These are generically referred to as deriving from C F U - G E M M . 7 3 However, for routine purposes the recognition of erythroid and granulopoietic elements apparently emanating from a common progenitor is usually the basis of designating the progentior as a C F U - G E M M . The recognition of colonies derived in vitro from the proliferation of a cell with stem cell characteristics (ie. high proliferative potential and self-renewal capability) began with murine hemopoiesis. Murine marrow cells that generated colonies within 14 days that were large enough to be visible macroscopically and that were red because they contained large numbers of hemoglobinized erythroblasts, were derived from a cell termed " B - m a c r o " . 7 4 These colonies were also found to usual ly contain megakaryocytes and g r a n u l o c y t e s 7 4 and many also contained C F U - S . 3 5 U p o n replating, many of these primary macroscopic multi-lineage colonies also gave rise to secondary macroscopic multi-lineage colonies of the same size and in 12 some Instances tertiary colonies could be o b t a i n e d . / 0 Subsequent studies revealed that some murine progenitors after 2 weeks had only produced small colonies (<1000 cells) still composed of cells with blast m o r p h o l o g y . 7 6 O n replating, 80% of the cells in these blast colonies gave rise to secondary colonies, many of which were of the large multilineage t y p e . 7 6 Analysis of the numbers and types of clonogenic progeny generated by individual primary colonies has provided important information about the heterogeneity i n proliferative and differentiative potential expressed by individual multipotent progenitors st imulated to divide under apparently identical conditions. 7 5 The most primitive type of human clonogenic progenitor currently recognized is the "blast colony-forming cell". However, exclusive assays for such cells have been difficult to devise because almost any normal hemopoietic colony that will ultimately contain more than a hundred cells will appear as a colony of blasts if evaluated at a sufficiently early stage in its development. The term, blast colony, in the context of assays of normal cells is therefore conventionally restricted to refer to colonies that are found to consist of blasts at a time when most types of colonies have (or, if not present, would have) already begun to reach maturity. Reliable detection of blast colonies requires that the starting population and the culture conditions be manipulated so as to drastically reduce the formation of colonies from the majority of myeloid progenitors with in vitro clonogenic p o t e n t i a l . 7 7 , 7 8 Two types of assays have been devised to achieve this. One involves plating cells in low concentrations of F C S with the delayed (2 weeks) addition of growth factors. The other involves culturing the cells in an agar layer overlying a stromal layer without added growth factors. Use of the former assay has documented the existence of a primitive cell type in human marrow that can be stimulated in vitro for 3 to 4 weeks to generate daughter clonogenic c e l l s . 7 7 The ability to recognize these colonies has allowed the definition of novel factor requirements of primitive hemopoietic c e l l s . 7 ^ ' 8 ^ However, neither human nor analogous murine blast colony-forming cells have 13 been shown conclusively to possess in vivo lympho-myeloid reconstituting potential, hence their precise position in the hemopoietic cell hierarchy is still unclear. C. Long-Term Bone Marrow Cultures Long-term bone marrow cultures ( L T C ) ® 1 represent a system i n which primitive hemopoietic progenitors associate with a layer of supportive adherent stromal cells to allow hemopoiesis to be maintained for many weeks in the absence of exogenously added growth factors. The adherent stromal cell components are derived from nonhemopoietic cells present in the bone marrow and include cells with features of endothelial cells, fibroblasts, adipocytes and smooth muscle cells. 12,82,83 I n M U R M E LTC the presence of such cells has been shown to be essential to obtain sustained h e m o p o i e s i s , ® 4 although the extent to which the various differentiated mesenchymal phenotypes observed in primary marrow cultures have individual roles is not known. The fact that i n murine L T C they can be effectively replaced by cloned, immortalized fibroblastoid cell lines suggests that a complex mixture of phenotypes may not be as i m p o r t a n t as c e r t a i n f e a t u r e s they m a y s h a r e , eg, i n t e r m s of g r o w t h f a c t o r p r o d u c t i o n . 8 5 " 8 7 H u m a n L T C can be established by suspending bone marrow cells with or without red cell a n d / o r granulocyte depletion i n sufficient numbers i n a suitable growth medium (eg, 3 x 1 0 6 nucleated cells per ml of L T C medium per 3 c m 2 tissue culture dish s u r f a c e ) . 8 1 , 8 8 Under these conditions stromal cell precursors in the original bone marrow suspension adhere to the bottom of the dish and proliferate to eventually form a confluent feeder layer. Alternatively, a source of primitive hemopoietic cells may be placed on a pre-established layer of adherent marrow c e l l s . 8 9 The presence of a pre-established feeder has no effect on the subsequent hemopoietic activity of cultures initiated with a large number of marrow cells (C.J. Eaves, personal communication), but has been used to allow expression of the hemopoietic potential of cell suspensions that contain inadequate numbers of stromal cells 14 or their precursors, y analogous to an optimal exogenous source of soluble growth factors in standard clonogenic cell assays. Functionally adequate, reproducible, feeders can be obtained by subculturing confluent adherent layers from 3 to 6 week old primary human marrow long-term cul tures . 8 9 In the mouse it was shown early on that most primitive cells become localized within the adherent l a y e r 9 0 within the first 24 hours of c u l t u r e . 9 1 In human L T C , when the adherent layer of these cultures is suspended by trypsinization, the majority of primitive clonogenic progenitors as defined by their large size are also found to be present in the adherent l a y e r . 9 2 Culture conditions that allowed the long-term maintenance of hemopoiesis in vitro (> 4 weeks) were first developed for murine bone marrow c e l l s . 9 3 , 9 4 Initially difficulties were encountered in two areas; only certain pre-selected batches of horse serum seemed to support hemopoiesis, and even with satisfactory batches of horse serum considerable inconsistency was e n c o u n t e r e d . 9 5 The latter problem was resolved first by the finding that cultures maintained at 3 3 ° C rather than 3 7 ° C consistently produced more C F U - S , C F U - G M , B F U - E and mature granulocytes for long periods of t i m e . 9 3 , 9 6 Addition of hydrocortisone to the culture m e d i u m 9 4 made selection of horse serum easier and thereby made the technology routinely available. Both of these changes also made it possible to obtain long-term maintenance of h u m a n hemopoiesis (for > 8 weeks) using the murine L T C protocol with selected batches of fetal calf and horse s e r u m . 8 8 In L T C initiated with murine bone marrow, cells with long-term in-vivo reconstituting ability have been shown to be maintained and for many w e e k s . 9 7 More recently, retroviral marking has been used to demonstrate that some of these cells have lymphoid as well as myelopoietic reconstituting potential and that they proliferate during their maintenance in L T C . 9 8 In LTC initiated with human bone marrow, cells with at least short-term repopulating 15 ability are likely to be present after 10 days of culture as shown by the prompt reconstitution of hemopoiesis i n patients given marrow ablative doses of chemoradiotherapy and a cultured marrow autograft .^.lOO T h u s > the unique feature of the LTC system over standard clonogenic assays is that it appears better able to support the maintenance and proliferation of a hemopoietic cell which is distinct from and more primitive than the cells detected by clonogenic assays. Characterization of the molecular mechanisms by which this is achieved is obviously of considerable interest. Both murine and human studies suggest that the cell with in vivo reconstituting abilities shares many characteristics with the cell responsible for the output of clonogenic progenitors after several weeks in L T C . ^ 1-105 F o r example studies of marrow cells exposed in vitro to 4-hydroperoxycyclophosphamide (4-HC), a cycle specific chemotherapeutic agent related to cyclophosphamide, have provided evidence that in humans as well as in rodents, most if not all clonogenic cells differ from cells capable of regenerating hemopoiesis in vivo by the much lower sensitivity of the latter type of cells to this drug. 104- 106 H u m a n marrow cells capable of initiating prolonged hemopoiesis in the L T C system, have also been found to have a similar insensitivity to 4 - H C . 1 0 2 , 1 0 7 These studies suggest that in L T C the clonogenic progenitors initially present terminally differentiate within the first 4 weeks, and that the clonogenic progenitors detected after 4 weeks are the result of the proliferative activity of a clonogenic cell precursor . Hereafter, this cell wil l be referred to as a " L T C - i n i t i a t i n g cell" (LTC-IC) . E x p e r i m e n t s wi th mouse marrow have shown that both m u r i n e L T C - I C a n d i n vivo repopulating cells are rhodarnine-123 dull, whereas all day 8 C F U - S and the majority of day 12 C F U - S are rhodamine b r i g h t . 3 7 ' !01 in human marrow, clonogenic cells and LTC-IC have been found to differ in their expression of CD33 ( M y 9 ) . 1 0 8 Initiation of sustained hemopoiesis in the L T C human marrow system would thus appear to offer a procedure for detecting cells that give rise to clonogenic progenitor cells but which may, themselves, be distinct from most clonogenic cells and therefore developmentally more closely related to repopulating cells. 16 Quantitative assessment of hemopoiesis i n L T C may thus depend upon the endpoint used. Measurement of the content of nonadherent mature clonogenic progenitors that are in the cells removed at the time of the weekly half-media changes is possible without disruption of the culture. However, such nonadherent cell measurements yield data that is difficult to interpret since the relative contributions of suriviving input clonogenic cells and newly produced clonogenic cells are likely to be continuously varying at least during the first 4 weeks of culture. Additionally, the proportion of clonogenic cells that are present in the nonadherent as opposed to the adherent fraction is also changing rapidly during the first few weeks. Initially, all cells are in the nonadherent fraction (the input), but after 2 weeks the majority of primitive clonogenic progenitors in the adherent l a y e r . 9 2 Thus exclusive assessment of the nonadherent layer, even at later times provides an assessment of hemopoiesis in this system that is both partial and biased towards the more mature clonogenic cell types and their granulopoietic progeny. To obtain a quantitative assessment of a more primitive cell it appeared that a better endpoint might be the number of clonogenic progeny in the entire LTC (adherent plus nonadherent cells) after 4 to 8 weeks of culture. The LTC-IC assay described in this thesis (figure 1) was thus based on the use of this endpoint. 17 BM Cells Total Clonogenic Cells Per LTC (Adh + NA Cells) | S1 Dishes with 33°C j Pre-established U^sj weekly 1/2 med. change LTC Adh Layers • , (+15 Gy) 0 1 2 3 4 5 weeks in culture FIGURE 1: Assay for LTC-IC. Unsorted or sorted human bone marrow cells were suspended In 2.5 ml of LTC media and plated on pre-established irradiated (15 gray) normal marrow adherent cells. Cultures were maintained at 37°C for 3 to 4 days then switched to 33°C in 5% CC<2. Half the media along with half the nonadherent cells were removed weekly and fresh media equal to the volume removed was returned to the cultures. After 5 weeks, all nonadherent cells were removed and adherent cells suspended by trypsinization. Nonadherent and adherent cells were plated separately in methylcellulose for assessment of the total clonogenic progenitor content of the LTC. 18 3.PURIFICATION O F PRIMITIVE H U M A N HEMOPOIETIC CELLS A. Physical separation techniques Isolation of stem cells from the majority of the nucleated bone marrow cells requires the identification of characteristics of the stem cells that differ from those of some or all or the other cells. The characteristics of cells that have previously been found to be useful for cell separation studies of marrow subpopulations include physical or structural properties such as density and size, functional properties such as adherence to plastic or phagocytic ability, and the cell surface antigen expression detectable by staining with monoclonal antibodies. Once the characteristics of the cell of interest are identified, these may then be used for preparative separation strategies. In some cases a simple physical separation is possible, such as using plastic adherence to deplete monocytes. In other cases separation is not necessary, as the non-desired cells are killed (for example using complement-fixing monoclonal antibodies plus fresh complement). Separation of cells according to surface antigen expression can be accomplished by a variety of methods. A surface, such as beads which are then loaded into a c o l u m n , 1 0 9 or a petri d i s h , c a n be coated with an ant ibody that recognizes mouse immunoglobulin. Any cell which expresses the relevant epitope on its surface will have bound the specific mouse monoclonal antibody and will thus be recognized by the anti-mouse immunoglobulin and be bound to the surface. This principle can be used both to remove nonreactive or negative cells from the bound positive cells or visa versa. The other common method for cell separation on the basis of either physical or surface antigen expression characteristics is cell sorting using a fluorescence activated cell sorter. Density separation has been used for many years to prepare h u m a n blood and bone marrow specimens for clonogenic assays, primarily as a way of reducing the number of red blood cells present as these interfere with colony visualization. Because of this, the density of 19 clonogenic cells has been studied extensively. Ficoll, metrizamide, albumin, and Percoll, a modified colloidal silica, have all been used for the isolation of subpopulations of hemopoietic cells. Using a discontinuous Percoll gradient, clonogenic cells have been found to peak at densities of 1.062 - 1.064 g / m l 1 1 0 " 1 1 2 and at 1.064 - 1.066 g / m l . 1 1 3 Using continuous Ficoll/Isopaque gradients, Francis demonstrated a difference between C F U - G E M M density (peak at 1.060 g / m l - range 1.055 - 1.064 g/ml) and C F U - G M density (peak at 1.064 g / m l -range 1.060 - 1.070 g / m l ) . 1 1 4 The density of LTC-IC has not been studied, however density separation has been used to prepare donor bone marrow or peripheral blood stem cells prior to transplantation, primarily to remove T cells from the graft as a way of preventing graft-vs-host disease or to reduce the volume and red blood cell contamination of the graft. The majority of patients sustained a successful engraftment after donor marrow had been density separated to remove cells with a density > 1.070 g /ml using Percoll followed by counterflow e lut r ia t ion . 1 1 5 The fluorescence activated cell sorter (FACS) is an instrument that allows the accurate analysis and sorting of cell populations on the basis of their ability to influence a light signal. Individual cells are analysed inside a capillary surrounded by sheath fluid. The F A C S used for the work described in this thesis has an argon ion L A S E R (Light Amplification by Stimulated Emisson of Radiation), a strong 488nm beam generated by the ionization of argon by a high voltage pulse. Deflection of the laser beam is measured at both 0 ° (forward light scatter (FLS)) using a photodiode and 9 0 ° (orthogonal light scatter (OLS)) using a photomultiplier tube. Although correlation with cell characteristics measured by more conventional methodology is not absolute, FLS is generally increased in cells with larger size, and OLS is increased in cells with high granularity or internal structure. Constant signal amplification can be maintained for analysis of different human blood and bone marrow samples by calibation of the instrument prior to each analysis with beads that give a signal of fixed intensity. 20 The ability of the F A C S to identify cell surface antigen characteristics of cells relies on the use of fluorochromes that can be directly or indirectly linked to monoclonal antibodies w h i c h recognize specific epitopes on var ious molecules present at the cell surface . Fluorochromes are so-named because they emit light at a longer wavelength than that which was used for their excitation. Different fluorochromes may emit signals of different wavelength despite similar excitation spectra. For example, while both fluorescein isothiocyanate (FITC) and phycoerythrin (PE) can be excited by 488nm light, FITC emits light maximally at 500 to 550 nm whereas PE emits light maximally at 550 to 600 nm. Fluorescent signal emission from individual cells is measured by the FACS through the use of a photomultiplier tube to amplify the signal, and the use of appropriate filters to detect light of the desired wavelength(s). The use of m o n o c l o n a l antibodies l inked to different f luorochromes c a n t h u s be used to simultaneously analyze individual cells for the expression of several antigens. Depending on the design of the F A C S , 3 or more parameters can be analyzed in total, two of which are usually FLS and OLS characteristics. As cells pass through the laser a sort decision is made prior to droplet formation depending upon the criteria established by the operator. If a cell is to be sorted, a voltage is applied to the fluid stream at a time when the droplet containing that cell is forming, such that the droplet is charged and can thus be deflected by two deflecting plates which carry a high voltage of opposite polarity. The droplet containing the selected cell is then deflected into a collecting tube to the right or left of the main stream which is itself directed into a central c o n t a i n e r . 1 1 6 B. Light scatter properties of primitive cells Normal h u m a n blood and bone marrow cells are heterogeneous in their light scatter properties as assessed by flow cytometry. The light scatter properties of many different cell types have been d e t e r m i n e d by sor t ing cel l p o p u l a t i o n s a n d t h e n a n a l y s i n g t h e m morphologically (figure 2). Platelets and cell debris have very low FLS and OLS characteristics. 21 Mature red blood cells and lymphocytes both have low OLS properties, but mature red blood cells have lower F L S characteristics. The "lymphocyte" window is seen clearly i n normal peripheral blood. In bone marrow a population with slightly higher OLS but the same FLS as the l y m p h o c y t e s c o n t a i n s n u c l e a t e d e r y t h r o i d ce l ls , p r i m a r i l y o r t h o c h r o m i c a n d polychromatophilic normoblasts. The "blast" window contains cells with a slightly higher FLS and OLS as compared to the lymphocytes and includes monocytes, myeloblasts, promyelocytes and some very early erythroid cells. The "granulocyte" window is in a region of relatively high OLS, with FLS slightly lower than that of the "blast" window. This region contains mature and nearly mature granulocytic cells. A continuum exists between the "blast" and "granulocyte" regions in bone marrow that includes maturing granulocytic cells such as myelocytes. 1 1 7 "121 22 ,vvvs granulocyte window ; red blood i cell window lymphocyte window [blast window 4 0 0 8 0 0 1200 FLS F I G U R E 2 A : L i g h t scat ter propert ies of n o r m a l h u m a n p e r i p h e r a l b l o o d . A 10% c o n t o u r h i s t o g r a m of F L S a n d O L S for f i c o l l e d p e r i p h e r a l b l o o d i s p r e s e n t e d . T h e l i g h t s c a t t e r of l y m p h o c y t e s ( " lymphocyte w i n d o w " ) , g r a n u l o c y t e s ( "granulocyte w i n d o w " ) , m o n o c y t e s ("blast window") , a n d r e d b l o o d cel ls i s presented. 400 8 0 0 1200 FLS granulocyte window red blood cell window lymphocyte window s== blast =1 window ... nucleated erythroid precursors F I G U R E 2 B : L i g h t s c a t t e r p r o p e r t i e s of n o r m a l h u m a n b o n e m a r r o w . L o w d e n s i t y (< 1 .068 g m / m l ) bone m a r r o w cel ls are a n a l y s e d i n a 10% c o n t o u r h i s t o g r a m of F L S a n d O L S . T h e l ight s c a t t e r r e g i o n s r e p r e s e n t i n g t h e m a j o r p o p u l a t i o n s of c e l l s r e p r e s e n t e d i n t h i s t i s s u e are presented. 23 The light scatter properties of clonogenic progenitors have also been investigated previously a n d used to obtain enriched populat ions of these cells , either alone or i n combination with other cell separation procedures. Beverley et a l 1 2 0 used high F L S i n combination with low expression of the human leukocyte common antigen (now known as C D 45) i n a population of normal human bone marrow cells depleted of mature myeloid cells by treatment with an anti-myeloid monoclonal antibody plus complement. The resulting populat ion was reported to contain 17% C F U - E , 3% C F U - G M , and 3% B F U - E , with an enrichment of C F U - G M of approximately 100-fold. Morstyn et a l 1 2 2 determined the light scatter properties of clonogenic cells by sorting 5 windows of both F L S and O L S . He determined their maximum concentration to be in a region of high FLS and low to intermediate OLS, i.e. in the "blast" window. Using FLS alone, a 10-fold enrichment in clonogenic cells was obtained. Using OLS alone, a 2- to 3-fold enrichment was achieved. Using both of these parameters p lus intermediate fluorescence for a fucose-binding lectin, a 20- to 30-fold enrichment was achieved. The location of the majority of the clonogenic cells i n the"blast" window has been subsequently confirmed by other i n v e s t i g a t o r s . 1 1 7 " 1 1 9 The light scatter properties of LTC-IC had not been previously reported, however coincident with publication of the work reported in Chapter 3 of this t h e s i s , 1 2 3 Andrews et al also described the light scatter properties of LTC-IC using a 4-week e n d p o i n t . 1 2 4 C. Use of anti-CD 34 monoclonal antibodies The first a n t i - C D 34 antibody produced was My-10 which was raised i n a mouse following immunization with the immature human myeloid leukemia cell line K G - l a . 1 2 5 This antibody was shown to bind to a 115,000 dalton glycoprotein of as yet unknown function, but expressed on only 1 - 4% of all normal human bone marrow c e l l s . 1 2 6 The marrow cells that do express C D 34 are mostly immature marrow cells (87% are blast cells by morphology) 1 2 5 and 24 Include virtually all clonogenic c e l l s . 1 2 6 , 1 2 7 The most primitive types of clonogenic cells such as C F U - G E M M and blast-colony-forming cells show on average a higher expression of C D 34 than do C F U - G M and B F U - E , and more mature cells such as C F U - E have a lower and more variable expression of C D 3 4 . 1 2 6 C D 34 is also expressed on the blasts of some patients with acute l e u k e m i a . 1 2 5 Subsequently, other anti-CD 34 antibodies were d e v e l o p e d . 1 2 8 - 1 3 1 One of t h e s e , 1 2 8 has been used to enrich for primitive hemopoietic cells in both baboon and human m a r r o w . 1 0 3 , 1 3 2 Bone marrow populations enriched for C D 34-positive cells successfully repopulated lethally irradiated baboons, whereas populations depleted of C D 34-positive cells did not, suggesting that the repopulating stem cell i n baboons is C D 34-positive. A few patients have recovered following lethal irradiation and infusion of autologous C D 34-positive cells suggesting that human repopulating cells may also be C D 3 4 - p o s i t i v e . 1 0 3 A significant advance i n the investigation of stem cells was recently provided by the development of a high affinity C D 34 monoclonal antibody, 8G12, which, unlike previously developed anti-CD 34 antibodies, can be directly coupled to fluorochromes thus providing a means for more sensitive multicolor analysis of C D 34-positive subpopulat ions . 1 3 1 C D 34 antibodies have been used both a l o n e , 1 2 6 , 1 2 8 and in combination with other monoclonal a n t i b o d i e s 1 2 4 , 1 3 3 to enrich for hemopoietic progenitors. When C D 34-positive cells from normal human bone marrow are sorted by FACS the resulting sorted population is 5 to 20% clonogenic p r o g e n i t o r s . 1 2 6 , 1 2 8 D. Other monoclonal antibodies used for the purification of primitive hemopoietic cells The majority of antigens found on bone marrow or blood cells recognized by currently available monoclonal antibodies do not appear to be expressed on hemopoietic progenitor cells. Indeed, efforts to purify stem cells i n the past have often focussed on the use of panels of monoclonal antibodies that recognize antigens on non-progenitor cells in depletion protocols, 25 e.g. using complement, physical separation techniques or FACS to remove the labelled cells. 1 3 4 ~ 1 3 8 In experiments by Griffin et a l , 1 3 4 such a depletion strategy was combined with positive selection for cells expressing HLA-DR antigen in the purification of clonogenic progenitors from the peripheral blood from patients with chronic myelogenous leukemia (CML). This resulted in a 50- to 100-fold enrichment, producing a population with 10 to 15% clonogenic cells and an additional 15 to 35% small cluster-forming cells. When a similar protocol was used to enrich for clonogenic cells in normal human bone marrow an 18-fold enrichment was achieved and the resultant population was -2% clonogenic c e l l s . 1 3 5 Using a panel of monoclonal antibodies with a negative selection protocol alone, Mouchiroud et a l 1 3 6 enriched clonogenic cells in normal human bone marrow 6- to 16-fold. When the same panel was used and cells not recognized by the panel of monoclonal antibodies were sorted in combination with appropriate light scatter gates, a population that was 5 to 10% clonogenic cells was obtained. With a panel of 10 lineage-specific monoclonal antibodies and a panning technique, Emerson et a l 1 3 4 enriched clonogenic progenitors from human fetal liver 100-fold resulting in a population of 30% BFU-E, 5% CFU-GM and 1.4% CFU-GEMM. Selection of cells that express the HLA-DR antigen has been used to enrich for clonogenic cells, as most of these cells have been shown to express this antigen. 139.140 when low density, nonadherent, T cell-depleted, bone marrow cells were sorted for high expression of CD 34 and low expression of HLA-DR (ie. the lower half of the HLA-DR-positive fraction of cells) a population with up to 47% clonogenic cells was achieved. 1 3 3 The reported expression of HLA-DR on more primitive cells has been more variable. The "blast colony-forming cell" was reported to lack surface HLA-DR antigen, 1 41 while LTC-IC have been reported to both express 1 4 0 and l a c k 1 4 2 , 1 4 3 HLA-DR antigens. Part of this controversy may be related to the possible use of different criteria to define HLA-DR negativity. 26 The C D 33 antigen, identified by monoclonal antibodies My 9 or L 4 F 3 , 1 0 8 has very recently been used to separate directly clonogenic cells from their precursors as assayed in the LTC-IC a s s a y . 1 2 4 Both unipotent and multipotent clonogenic cells express C D 33 while L T C -IC do n o t . 1 0 8 , 1 2 4 Other n o n - C D 34 monoclonal antibodies have been found to react with hemopoie t i c progeni tors a n d some l e u k e m i c cel ls i n p a t i e n t s w i t h acute m y e l o i d l e u k e m i a . 1 1 9 , 1 3 8 , 1 4 4 , 1 4 5 some of these have also been used successfully for obtaining enriched populations of primitive hemopoietic cells. 4. GROWTH FACTOR REGULATION OF HEMOPOIESIS A.Colonv-stimulating factors The development of semi-solid assays for myeloid progenitors has allowed the isolation of a number of molecules, originally found in media conditioned by various cell types, that are required to support the formation in vitro of different types of hemopoietic colonies. These activities were originally called colony-stimulating factors (CSF). This name has been retained for 3 such factors that have been subsequently purified and shown to represent the products of distinct genes. One of these, CSF-1, is a molecule found to stimulate primarily macrophage (M) colonies i n assays of mouse marrow and has therefore also been called M - C S F . I 4 6 Another molecule originally found to stimulate primarily granulocytic (G) colonies is called G - C S F . I 4 7 A molecule found to stimulate both granulocyte and macrophage colonies as well as colonies containing both of these cell types has been called G M - C S F . i 4 8 CSF-1 is a hematopoietin that is best documented for its ability to support the survival, proliferation and differentiation of cells of the mononuclear phagocyte series, particularly in the murine system. I 4 6 When given as a single injection to rats in vivo it resulted i n a dose-dependent increase i n peripheral monocytes and a slight increase i n neutrophils with an 27 associated decrease i n the lymphocytes i n the peripheral b l o o d . 1 4 9 The molecule is a heavily glycosylated homodimeric protein with a molecular mass of -70,000 daltons and is encoded by a single gene of about 18 kb that maps to the long arm of human chromosome 5 at band 3 3 . 1 . 1 5 0 , 1 5 1 By alternative splicing of messenger RNAs either secreted or membrane bound forms are produced. 1 5 1 CSF-1 is produced constitutively by fibroblasts, endothelial cells and m o n o c y t e s 1 0 - 1 5 2 and CSF-1 activity can be detected in s e r a . 1 5 3 It has been postulated that serum CSF-1 levels are controlled by macrophages themselves, as these cells can specifically clear the growth factor by CSF-1 receptor mediated e n d o c y t o s i s . 1 5 4 For many years it has been known that human CSF-1 can effectively stimulate murine progenitors but not vice v e r s a . 1 5 0 The receptor for CSF-1 is encoded by the c-frns protooncogene. 1 5 5 This receptor has tyrosine kinase activity and is a 972 amino acid glycoprotein encoded by a gene that also maps to human chromosome 5, at band 5q33.3. 1 5 0 Both a cloned murine myeloid progenitor cell line, 32D, and some immortalized murine pre-B-cell lines, when transfected to express c-fms, differentiated into macrophages when grown in the presence of human CSF-1 suggesting a possible deterministic role for this growth factor-receptor system. 156,157 G-CSF is a heavily glycosylated polypeptide of 207 amino acids (molecular mass -20,000 daltons) that is encoded by a gene found on chromosome 17 in the q l 1-12 region. 158-160 Both murine and human G-CSF can stimulate the target cells of the same or opposite species equally e f f e c t i v e l y . 1 6 1 Moreover, i n addition to stimulating the proliferation of mature granulocytic colonies, G-CSF can synergise with IL 3 to support blast colony formation in both s p e c i e s . 1 6 2 , 1 6 3 In vivo administration of G-CSF results i n a dose-dependent increase in functionally active neutrophils and is capable of shortening the neutropenic phase post-chemotherapy. 1 6 4 G-CSF has been shown to abrogate the periodic neutropenic episodes in dogs with the autosomal recessive disorder canine cyclic neutropenia. 1 6 5 Patients with cyclic neutropenia treated with G-CSF continued to have cycling of their blood counts, however neutropenic days were virtually e l i m i n a t e d . 1 6 6 In vivo administration of G-CSF to mice 28 resulted in an early (24 hour) increase in myeloblasts and promyelocytes in the bone marrow and G - C S F was synergistic in this regard with IL-3. However in neither case was there any change in overall bone marrow cellularity with the doses of growth factor u s e d . 1 6 7 G - C S F mRNA expression and production by monocytes is not constitutive but can be induced by lipopolysaccharide ( L P S ) . 1 0 mRNA expression and the production of bioactive material is induced by IL-1 in endothelial cells and fibroblasts . 1 0 > 1 1 • 1 5 2 Expression of G - C S F mRNA in the adherent layer of established h u m a n marrow L T C was not detected in unstimulated cultures, however after addition of fresh medium, IL- lp , or platelet derived growth factor (PDGF) mRNA increases up to 50 fold were f o u n d . 1 5 2 The receptor for murine G - C S F has recently been cloned and shown to be a member of the same receptor family (lacking intrinsic tyrosine kinase activity) to which many of the other hemopoietic growth factor receptors b e l o n g . 1 6 8 G M - C S F is also a glycoprotein, with a molecular mass of -22,000 daltons. The D N A sequence for human G M - C S F contains a 432 nucleotide open reading frame which encodes a 144 amino acid protein. 17 amino acids are cleaved from the precursor protein to yield a mature 127 amino acid mature f o r m . 1 6 9 Unlike CSF-1 and G - C S F , human G M - C S F cannot stimulate murine cells and murine G M - C S F does not stimulate human cells. The gene for human G M - C S F has been mapped to chromosome 5q21-32 by somatic cell hybrid analysis and in situ h y b r i d i z a t i o n . 1 6 0 G M - C S F stimulates some clonogenic cells of all myeloid lineages in v i t r o . 1 7 0 " 1 7 2 G M - C S F can also enhance neutrophil function by augmenting the cytotoxicity of these cells and by inhibiting their m i g r a t i o n . 1 7 2 In vivo, G M - C S F augments the peripheral neutrophil count and shortens the neutropenic phase post-chemotherapy. 1 7 3 It has therefore been used to treat patients with reduced or ineffective hemopoiesis such as occurs in aplastic anemia, myelodysplasia or acquired immunodeficiency syndrome, and in most such cases was successful in augmenting the peripheral neutrophil c o u n t . 1 7 4 ' 1 7 5 Increases in circulating clonogenic hemopoietic progenitor numbers have been reported post G M - C S F t h e r a p y . 1 7 6 In 29 the mouse, in vivo administration of G M - C S F has been reported to decrease bone marrow cellularity and absolute nonerythroid clonogenic cell n u m b e r s . 1 7 7 However, in primates, G M -C S F has been reported to increase bone marrow ce l l u l ar i t y . 1 7 3 A low affinity receptor for G M -C S F has been cloned, which is a 400 amino acid glycoprotein that lacks tyrosine kinase a c t i v i t y . 1 7 8 Very recently a second molecule also lacking a tyrosine kinase domain has been \ identified, which when co-expressed with the low affinity G M - C S F receptor converts it to a high affinity r e c e p t o r . 1 7 9 Erythropoietin stimulates erythropoietic differentiation, demonstrable in vitro by the support of colony formation by both C F U - E and B F U - E . Dose-response curves have indicated that murine C F U - E s t imulat ion is maximal at m u c h lower doses of erythropoietin by comparison to B F U - E , 1 8 0 with a similar trend suggested for h u m a n C F U - E and B F U - E . However B F U - E cannot be maximally stimulated by erythropoietin alone and require other factors such as IL-3, G M - C S F , IL-9, or the ligand for c - k i t . 6 4 - 1 8 0 " 1 8 2 Erythropoietin is produced in kidney tubules in response to hypoxia, as demonstrated by the upregulation of erythropoietin mRNA within 1 hour of the initiation of hypoxia in this tissue in a rat m o d e l . 1 8 3 Both erythropoiet in 1 8 4 and its r e c e p t o r 1 8 5 have been cloned and recombinant erythropoietin has been used therapeutically. The severe anemia associated with end stage renal disease is corrected by the administration of erythropoietin. 1 8 6 Most of the other factors now known to have hemopoietic colony-stimulating activity have been called interleukins because they are produced by one type of leukocyte and act on another. One of these that has been intensively studied is interleukin-3 (IL-3), also known by a variety of other names including "multi-CSF" because of its potent stimulating effects on the early stages of erythroid colony development from primitive erythroid cells, as well as on cells of many other l i n e a g e s . 1 8 1 Now that all of the CSFs as well as 11 interleukins and other factors active on hemopoietic cells are available as recombinant, purified reagents, it has become clear 30 that the actions of these factors are much less exclusive than often originally assumed. For example, G M - C S F and IL-3 show extensive, although not complete overlap on the cells they can s t i m u l a t e , 1 8 7 ' 1 8 8 possibly as a result of shared receptor structures, or receptor-associated molecules, or common downstream signal t ransduction p a t h w a y s . 1 8 9 ' 1 9 1 Additionally, many of these growth factors are also known to have nonhemopoietic activities. For example, CSF-1 is produced at high levels by the uterine epithelium during pregnancy and CSF-1 receptors are present on placental t r o p h o b l a s t s . 1 5 0 H u m a n G M - C S F stimulates the growth of a variety of transformed cell lines of nonhemopoietic o r i g i n , 1 9 2 and is also present on placental c e l l s . 1 6 9 . B. Interleukins H u m a n IL-3 was cloned using a gibbon T cell line to isolate a gibbon IL-3 cDNA clone, which was then used to isolate the human g e n e . 1 9 3 The recombinant protein is 20 to 26,000 daltons and contains 133 amino acids in its mature form. The human IL-3 gene is located on chromosome 5. Human IL-3 directly stimulates the formation of granulocyte colonies derived from eosinophil progenitors and to some extent also from neutrophil and macrophage progenitors. When combined with erythropoietin, IL-3 is a potent stimulant for colony formation by erythroid and multilineage progenitors . 1 8 1 IL-3 has also been shown to directly stimulate the formation of blast c o l o n i e s . 1 8 8 In vivo, in primates, IL-3 alone results in only a modest increase in leucocytes, primarily due to a b a s o p h i l i a , 1 9 4 but has an additive effect w h e n given with G - C S F . 1 4 9 W h e n given pr ior to or concomitant wi th G M - C S F or erythropoietin, IL-3 has a synergistic or priming effect in stimulating leucocytosis and reticulocytosis r e s p e c t i v e l y . 1 9 4 " 1 9 6 Like G M - C S F , human IL-3 and murine IL-3 are not cross-reactive between these 2 species. IL-3 has not been detected in serum in the steady state and is not produced by bone marrow stromal cells as determined by mRNA analysis and bioactivity assessment of stromal cells in vitro. 10.11.152 T lymphocyte production of IL-3 depends upon 31 the stimulation of these cells, such as by IL-1. Monocytes respond to LPS by the production of IL-1, and this cell may play a central role in stimulating the production of many CSFs . The murine IL-3 receptor has recently been c l o n e d . 1 9 7 A large number of other glycoprotein molecules with C S F activity on human or murine myeloid progenitors have also now been identified. These include IL-5,198,199 I L . g 200 I L _ 7,201 I L - 9 , 2 0 2 I L - 1 0 2 0 3 and I L - 1 1 . 2 0 4 Both m u r i n e l 9 8 and human 1 9 9 IL-5 stimulate the production of eosinophil colonies and this is augmented when this factor is combined with G M -C S F or IL-3. I L - 7 2 ° 1 stimulates the formation of murine B cell colonies but has not been found to stimulate the formation of granulocyte or macrophage colonies either alone or in synergy with other f a c t o r s . 6 3 However, it has been reported to selectively support megakaryocyte m a t u r a t i o n , 2 0 5 and it may stimulate stem cells, as it enhanced retroviral mediated gene transfer to murine repopulating stem cells, a procedure known to be enhanced by other growth f a c t o r s . 2 0 6 I L - 9 2 0 2 acts i n a dose-dependent manner to stimulate the proliferation of h u m a n peripheral blood and bone marrow B F U - E i n combination with erythropoietin. ! 8 2 This activity is maintained even with highly purified populations and under serum-deprived conditions suggesting that the action of IL-9 on these cells is direct. IL-10 can synergise with either IL-3 or IL-4 in stimulating the proliferation of murine mast cell l i n e s . 2 0 3 I L - 1 1 2 0 4 was isolated from a cDNA library prepared from a simian bone marrow stromal cell line. IL-11 stimulates the production of immunoglobulin-producing B cells and synergises with IL-3 in supporting murine megakaryocyte colony growth. IL-6 has also been called hybridoma growth factor, B cell growth factor-2, and interferon-P 2 2 0 7 because of its pleotropic activities, which include induction of the differentiation of B cells into antibody-producing cells, antiviral activity, induction of acute phase proteins in hepatocytes, and support for the proliferation of myeloma c e l l s . 2 0 8 IL-6 alone can stimulate the proliferation of murine C F U - G M and of blast cell c o l o n i e s . 2 0 9 When combined with IL-3, 32 blast cell colonies from bone marrow cells taken from 5-fluorouracil treated mice appeared earlier and in increased numbers as compared to what was observed with either growth factor a l o n e . 1 6 3 , 2 0 9 IL-6 also synergises with combinations of IL-3, IL-4, G-CSF, and erythropoietin to increase the number of granulocyte, macrophage, erythroid and megakaryocyte colonies produced from murine bone m a r r o w . 2 1 0 , 2 1 1 When IL-6 was administered to mice in vivo, increases in circulating neutrophils and platelets were demonstrated, as well as an increase in clonogenic progenitors ( C F U - G M and B F U - E ) i n the marrow and spleen of the treated a n i m a l s . 2 1 2 IL-6 is produced by monocytes, cultured endothelial cells and after I L - l p stimulation by bone marrow stromal c e l l s . 1 5 2 , 2 0 7 The receptor for IL-6 has also been cloned and belongs to the hemopoietic growth factor receptor f a m i l y . 2 1 3 In addition, a second associated protein, gp 130, probably responsible for signal transduction has also been c l o n e d . 2 1 4 The ability of various factors to synergize in stimulating various aspects of myelopoiesis in vitro has revealed the importance of testing combinations of factors which may alone not appear to have much effect. IL-1, IL-4 , IL-6, IL-7, IL-11, leukemia inhibitory factor, and the l i g a n d for c -ki t (see below) are examples of factors k n o w n to par t i c ipa te i n s u c h synergisms.64,80,163 5. STROMAL C E L L REGULATION OF HEMOPOIESIS The essential nature of the interaction between primitive hemopoietic cells and bone marrow stromal cells highlighted early on by the studies of Friedenstein et al in the 1960 ' s . 2 1 5 These studies showed that when syngeneic or semisyngeneic bone marrow tissue grafts were placed u n d e r the renal capsule of a mouse , recipient derived hemopoiesis c o u l d be demonstated several months later indicating that the transplanted bone marrow provided a 33 supportive environment for the implantation and differentiation of circulating host stem cells. It was subsequently shown that stromal cell precursors capable of forming fibroblast colonies are the cells responsible for the transfer of the microenvironment typical of hemopoietic tissue, and that differences in the tissue of origin (spleen vs marrow) of the stromal precursor resulted in differences in the relative proportions of different lineages of hemopoietic c e l l s . 2 1 6 In both murine and human L T C , hemopoiesis can be maintained for several months, and in the mouse this has been shown to depend on interactions that take place between primitive hematopoietic cells and marrow stromal cells that are present in the adherent layer of the c u l t u r e . 8 4 , 9 5 Close cell to cell interactions appear to be essential for the successful support of stem cells by the stromal microenvironment. 2 1 7 It is also possible that local retention of hemopoietic growth factors such as G M - C S F and IL-3 by binding to heparan sulfate produced by stromal cells may play an important role in hemopoietic cell regulation. 1 6 Primitive human stem cells have been shown to bind selectively to s t r o m a . 2 1 8 Evidence that the binding of murine stem cells to s troma may be dependent u p o n galactosyl and m a n n o s y l specificities has also been r e p o r t e d . 2 1 9 Mice with a congenital macrocytic anemia characterized by the S l / S l ^ (steel) mutation have normal hemopoietic cells in terms of their content of in vitro clonogenic cells and C F U - S , as well as their content of cells that can cure stem cell deficient W / W v mice. S l / S l ^ mice themselves, however, are not cured by injected suspensions of +/+ marrow cells, although they can be cured by transplantation of whole pieces of hemopoietic tissue. This suggests that there is a genetically determined defect in the hemopoietic microenvironment of S l / S l ^ m i c e . 2 2 0 , 2 2 1 This defect can also be transferred into the L T C system where a morphologically normal appearing feeder layer forms when S l / S l ^ marrow is used, but the output of hemopoietic cells is reduced. This abnormality can, however, be corrected by the use of pre-established feeders from normal or W / W v m i c e . 2 2 2 Thus both in the mouse and in L T C , stromal cells from the marrow appear to provide a unique supportive structure that is essential for the maintenance 34 of hemopoiesis. The functional specificity of, or requirement for, stromal cells of marrow origin is not yet clear as appropriately induced non-marrow derived mesenchymal cells appear also capable of creating a microenvironment in vivo that can support h e m o p o i e s i s . 2 2 3 Similarly, NIH 3 T 3 cells have been shown to be capable of supporting murine hemopoiesis in vitro in the L T C s y s t e m . 8 7 Recently, the stem cell abnormality in W / W v mice has been identified as a mutation in the c-kit gene which encodes a product with features of a c-fms-like growth factor 094. receptor . ' ' ^ The c-kit ligand has also recently been cloned and mapped to the steel locus, with mutations in this molecule identified to correspond to the defect in S l / S l ^ m i c e . 6 4 , 2 2 5 The c-kit ligand has been shown to synergize with other growth factors in the stimulation of many types of hemopoietic cells in vitro. Although the role of this factor in stromal cell mediated control of stem cell proliferation has not yet been fully clarified, it is of interest that one of the forms of the c-kit ligand expressed is a transmembrane (membrane bound) m o l e c u l e . 2 2 6 Since the extended hemopoiesis that is characteristic of L T C appears to depend on the presence of a feeder layer of mesenchymal cells, it seems likely that this role is mediated by the production of one or more factors with stimulatory/positive effects on primitive hemopoietic cells. In human marrow LTC, all progenitors in the nonadherent fraction of the culture, and the more mature, lower proliferative potential progenitors in the adherent layer remain in a continuously proliferating mode. In contrast the primitive (high proliferative potential) progenitors in the adherent layer and only in the adherent layer (where, however the majority of this progenitor type are located) must be stimulated to proliferate. This can be achieved by a change of the medium, but the stimulation so achieved is transient, and within a week the primitive clonogenic cells again return to a quiescent s t a t e . 2 2 7 More recently it was demonstrated that the substitution of cytokines such as IL-ip and PDGF for fresh media would mimic this stimulatory effect on primitive adherent layer progenitor c y c l i n g . 2 2 8 As these cytokines also cause an increase in mRNA for G-CSF, G M - C S F , IL-1 and IL-6 in stromal cells, 35 it is possible that stromal cell production of these (and/or other) hemopoietic growth factors is involved in the regulation of hemopoies is . 1 5 2 In fact, recent experiments in which G - C S F was added repeatedly to cultures, or cells were cultured on human bone marrow fibroblast feeders engineered to produce high levels of G-CSF, have shown that G - C S F can induce primitive cells to enter S-phase and can increase the total progenitor content of L T C after a period of 5 weeks. This suggests that G-CSF may stimulate primitive cells in LTC to induce their proliferation and differentiation. In contrast, neither G M - C S F addition, nor GM-CSF-producing feeders, was able to stimulate a similar effect on primitive progenitor cycling or progenitor numbers, although mature cell output (nonadherent cell numbers) was increased. Thus G M - C S F may have a greater selectivity for the later stages of hemopoiesis when this occurs in the presence of stromal c e l l s . 2 2 9 ' 2 3 0 In L T C of unmanipulated h u m a n marrow primitive progenitors adherent to stroma return to a quiescent state 1 week after a media change indicating that the stimulatory effect of the media change is transient and that there may be production of an inhibitor by the stromal cells. The ability of stromal cells to selectively arrest the cycling of primitive hemopoietic cells can be mimicked by the simultaneous addition of TGF-(3 at the time of media change which prevents the induction of cycling by fresh m e d i a . 2 2 8 TGF-(3 has been shown to be produced in LTC and addition of anti-TGF-p" antibody 3 days after a standard medium change can prevent primitive clonogenic progenitors in the adherent layer from returning to a quiescent state 4 days later, suggesting that stromal cells do, in fact, exert a negative effect on primitive hemopoietic cells, at least in part, by production of T G F - p \ 2 3 1 Thus, it appears likely that marrow stromal cells may provide important positive and negative regulatory signals for hemopoietic cells and also promote their survival. While hemopoietic growth factors such as IL-3, G - C S F and G M - C S F are essential for growth in 36 clonogenic cell assays and can influence hemopoiesis in the presence of stroma, their role in the normal regulatory mechanisms in the bone marrow remains unclear. 6. THESIS OBJECTIVES My work is based on the premise that, in order to understand basic mechanisms involved in the early control of hemopoiesis in the normal state, and hence to define molecular changes resulting in malignant transformation leading to leukemia, it was necessary to analyze hemopoiesis at the earliest stage of hemopoietic cell development, i.e. at the level of the hemopoietic stem cell. Since at the time I began these studies (in 1986) there were no known ways to identify this cell, the first objective of my research was to try to develop an assay that might prove suitable for the quantitation and characterization of more primitive cells than could be identified by standard colony assay procedures. Because there was some evidence that the cell responsible for the in i t ia t ion of long- term hemopoiesis i n L T C shared characteristics with in vivo repopulating cells, the LTC system appeared to offer an approach to this objective. O n the basis largely of the 4 - H C findings described above, production of clonogenic cells after 5 weeks was chosen as an endpoint to define a precursor cell which was then given the operational term of LTC-IC. Since it was anticipated that this cell would be present at very low concentrations in normal bone marrow (< 1%) which would contain many potential regulatory cells as well as more differentiated progeny, further analysis of LTC-IC regulation in vitro required that procedures be developed for its purification and separation from endogenous regulatory elements. Thus the first immediate objective of my research was to characterize the physical and surface antigen characteristics of stem cells and to use this information to develop the required purification procedures. At the same time, I also sought to validate the endpoint used in anticipation of then using limiting dilution techniques to quantitate absolute numbers of LTC-IC in a given population and to assess the proliferation 37 and differentiation of individual LTC-IC under standard LTC conditions. The final objective of my research was to begin to characterize the regulatory actions of stromal cells and specific growth factors on these cells. 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G o o d w i n R G , L u p t o n S, Schmierer A , Hjerrild K J , Jerzy R, Clevenger W, Gil l is S, Cosman D, Namen A E : Human interleukin 7: Molecular cloning and growth factor activity on human and murine B-lineage cells. Proc Natl Acad Sci U S A 86:302, 1989 202. Yang Y-C, Ricciardi S, Ciarletta A, Calvetti J , Kelleher K, Clark S C : Expression cloning of a cDNA encoding a novel human hematopoietic growth factor: Human homologue of murine T-cell growth factor P40. Blood 74:1880, 1989 203. Thompson-Snipes L, Dhar V, Moore K, Bond M , Rennick D : A T cell derived lymphokine termed cytokine synthesis inhibitory factor (CSIF) (interleukin-10) is a potent costimulant of mast cell growth. Exp Hematol 18:612, 1990 (abstr) 204. Paul SR, Bennett F, Calvetti J A , Kelleher K, Wood CR, O'Hara R M , Leary A C , Sibley B , Clark SC, Williams DA, Yang Y - C : Molecular cloning of a cDNA encoding interleukin 11, a stromal cell-derived lymphopoietic and hematopoietic cytokine. 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Rennlck D, Jackson J , Yang G, Wideman J , Lee F, Hudak S: Interleukin-6 interacts with interleukin-4 and other hematopoietic growth factors to selectively enhance the growth of megakaryocytic, erythroid, myeloid and multipotential progenitor cells. Blood 73:1828, 1989 211. Lotem J , Shabo Y, Sachs L: Regulation of megakaryocyte development by interleukin-6. Blood 74:1545, 1989 212. Pojda Z, Tsuboi A : In vivo effects of human recombinant interleukin 6 on hemopoietic stem and progenitor cells and circulating blood cells i n normal mice. Exp Hematol 18:1034, 1990 213. Y a m a s a k i K, H i r a t i a Y, Ywata H , K a w a n i s h i Y, Seed B , T a n a i g u s h T , H i r a n o T , Kishimoto T : Cloning and expression of the h u m a n interleukin-6 (BSF-2/IFNbeta2) receptor. Science 241:825, 1988 214. T a g a T , H i b i M , Y a m a s a k i K, Y a s u k a w a K, M a t s u d a T , H i r a n o T , K i s h i m o t o T : Interleukin-6 triggers the association of its receptor with a possible signal transducer, gp 130. Cell 58:573, 1989 215. Friedenstein A J , Petrakova KV, Kurolesova A l , Frolova GP: Heterotopic transplants of bone marrow. A n a l y s i s of p r e c u r s o r cells for osteogenic a n d hematopoietic t i ssues . Transplantation 6:230, 1968 216. Friedenstein A J , Chailakhyan RK, Latsinik NV, Panasyuk A F , Keiliss-Borok IV: Stromal cells responsible for transferring the microenvironment of the hemopoietic tissues. Transplantation 17:331, 1974 217. Bentley SA: Close range celhcell interaction required for stem cell maintenance in continuous bone marrow culture. Exp Hematol 9:308, 1981 218. Verfaillie C, Blakolmer K, McGlave P: Purified primitive human hematopoietic progenitor cells with long-term in vitro repopulating capacity adhere selectively to irradiated bone marrow stroma. J Exp Med 172:509, 1990 219. Aizawa S, Tavassoli M : Detection of membrane lectins on the surface of hemopoietic progenitor cells and their changing pattern during differentiation. Exp Hematol 16:325, 1988 220. Bernstein S E : Tissue transplantation as an analytic and therapeutic tool i n hereditary anemias. A m J Surg 119:448, 1970 53 221. McCulloch EA, Siminovitch L, Till J E , Russell ES , Bernstein SE : The cellular basis of the genetically determined hemopoietic defect in anaemic mice of genotype S l / S l ^ . Blood 26:399, 1965 222. Dexter T M , Moore M A S : In vitro duplication and "cure" of haemopoietic defects in genetically anaemic mice. Nature 269:412, 1977 223. Reddi A H , Huggins CB: Formation of bone marrow in fibroblast - transformation ossicles. Proc Natl Acad Sci USA 72:2212, 1975 224. Chabot B, Stephenson DA, Chapman V M , Besmer P, Bernstein A: The proto-oncogene c-kit encoding a transmembrane tyrosine kinase receptor maps to the mouse W locus. Nature 335:88, 1988 225. Martin F H , Suggs SV, Langley K E , L u HS, Ting J , Okino K H , Morris C F , McNiece IK, Jacobsen FW, Mendiaz EA, Birkett N C , Smith KA, Johnson M J , Parker VP, Flores J C , Patel A C , Fisher E F , Erjavec H O , Herrera C J , Wypych J , Sachdev RK, Pope JA, Leslie I, Wen D, Lin C - H , Cupples RL, Zsebo K M : Primary structure and functional expression of rat and human stem cell factor DNAs. Cell 63:203, 1990 226. Flanagan J G , Leder P: The kit ligand: A cell surface molecule altered i n steel mutant fibroblasts. Cell 63:185, 1990 227. C a s h m a n J , Eaves A C , Eaves C J : Regulated proliferation of primitive hematopoietic progenitor cells in long-term human marrow cultures. Blood 66:1002, 1985 228. Cashman J D , Eaves A C , Raines EW, Ross R, Eaves C J : Mechanisms that regulate the cell cycle status of very primitive hematopoietic cells in long-term human marrow cultures. I. Stimulatory role of a variety of mesenchymal cell activators and inhibitory role of TGF-(3. Blood 75:96, 1990 229. Hogge D E , Cashman J D , Humphries RK, Eaves C J : Differential and synergistic effects of human granulocyte-macrophage colony stimulating factor and human granulocyte colony stimulating factor on hematopoiesis in human long-term marrow cultures. Blood 77:493, 1991 230. Coutinho L H , Will A, Radford J , Schiro R, Testa NG, Dexter T M : Effects of recombinant human granulocyte colony-stimulating factor (CSF), human granulocyte macrophage-CSF, and gibbon interleukin-3 on hematopoiesis in human long-term bone marrow culture. Blood 75:2118, 1990 231. Eaves C J , C a s h m a n J D , Kay R J , Dougherty G J , Otsuka T, Gaboury LA, Hogge D E , Lansdorp PM, Eaves A C , Humphries RK: Mechanisms that regulate the cell cycle status of very primitive hematopoietic cells in long-term human marrow cultures. II. Analysis of positive and negative regulators produced by stromal cells within the adherent layer. Blood (in press) 54 C H A P T E R II MATERIALS AND M E T H O D S 1. C E L L S (A) Bone Marrow Cells H u m a n bone marrow was obtained from informed and consenting individuals donating marrow for autologous or allogeneic marrow transplants after approval of the Cl inica l Screening Committee for Research Involving Human Subjects and was collected in TCI99 with 100-200 U / m l of preservative free heparin and stored on ice until processed. In some cases a marrow buffy coat was prepared using an aliquot of the marrow sample. The sample was centrifuged at 200 g for 4 minutes and the buffy coat layer was removed from the interface between the plasma and the red blood cells using a Pasteur pipette. A low density bone marrow cell fraction was isolated on the same day of the bone marrow collection, using a modification of the methods of F r i c k h o f e n 1 and Roos and de B o e r 2 for discontinuous Percoll density centrifugation. A n isosmotic Percoll stock solution was prepared by mixing Percoll (Pharmacia Fine Chemicals, Uppsala, Sweden) with a 10X concentrated phosphate-buffered saline solution to obtain a Percoll stock solution with a measured osmolality of 290 ± 5 m O s m and a pH of 7.4. Marrow cells were washed twice i n Iscove's medium with 2% fetal calf serum (FCS), suspended in the same at 1 0 7 cel ls /ml , a microhematocrit determined, and the cells then mixed with an appropriate volume of Percoll to suspend the cells in a solution with a density of either 1.066 g / c m 3 or 1.068 g / c m 3 , as follows: 55 Volume of Percoll Stock Density Desired (1.066 or 1.068 g/cm ; Density of Iscove's 2% F C S Volume x of Cells Hematocrit TDD Solution to be Added (ml) = Density ot Percoll Stock Solution Desired Density & Wash (ml) x 1 -The suspension was overlayered with 5 ml of Iscove's with 2% F C S and centrifuged at 600 g for 15 minutes at 22pC. The cells floating on top of the Percoll solution were removed and washed twice in Iscove's with 2% F C S . Percoll separated cells were stored at 4°C overnight in Iscove's with 30% F C S prior to staining and sorting. Initial experiments were performed with cells with a density < 1.066 g / c m 3 , but for most experiments, a slightly higher density cut-off of 1.068 g / c m 3 was used. Nucleated cell counts were performed on a Coulter Counter Model Z M (Coulter Electronics Limited, Luton, England). (B) Cell Line Maintenance *¥2 ce l ls 3 and NIH-3T3 cells were cultured in Dulbecco's modified Eagles medium with high glucose (4.5 g/L) and 10% heat-inactivated calf serum (for *P2 cells) or 10% F C S (for NIH-3T3 cells). M2-10B4 cells, a cloned murine (B6C3Fj) marrow fibroblast cell l i n e 4 were maintained in RPMI medium plus 10% F C S . AML-193 cells (ATCC, Rockville, MD) were grown in Iscove's medium with 20% FCS and 10% medium conditioned by the 5637 cell line (American Type Culture Collection, Rockville, MD) as a source of human growth factors. 5 NFS-60 cel ls 6 were grown in RPMI with 20% FCS and 5% pokeweed stimulated mouse spleen cell conditioned m e d i u m . 7 B9 cells were grown in D M E M with 10% F C S and 100 U / m l of recombinant IL-6 obtained from Dr. P. Lansdorp. 8 56 2. STAINING AND FLOW CYTOMETRY (A) Studies of Light Scatter, H L A - D R and C D 34 Reactivity of Clonogenic Cells and LTC-IC  (Chapters III, V.VI) Low density cells were washed twice in Hanks' buffered saline with 2% F C S and 0.1% sodium azide (HFN), suspended in H F N at 5 x 1 0 6 cells/ml with 2 u.g/ml propridium iodide (PI) and 100 ug/ml DNase I (Type II-S, Sigma Chemical Co., St. Louis. M O , U.S.A.) and sorted on the basis of their light scatter properties. For labelling with (fluorescent) antibodies, low density cells were washed and suspended in H F N at 2 x 10 7 cells/ml. Cells to be stained with anti-CD 34 (HPCA-1, Becton Dickinson, Sunnyvale, CA, U.SA.) were incubated with 2 ug of antibody per 1 0 7 cells for 30 m i n u t e s on ice, washed with H F N , a n d then stained with f luorescein isothiocyanate (FITC)-conjugated F(ab)2 fragments of sheep anti-mouse IgG (SAM-FITC, Organon Teknika Corp., West Chester, PA) at 2 u.g of antibody per 10 7 cells for 30 minutes on ice. Cells were then washed again and suspended i n H F N with PI and DNase I. Cells stained with ant i -HLA-DR conjugated directly to phycoerythrin (PE) (anti -HLA-DR-PE, Becton Dickinson) were incubated with 1 u.g antibody per 1 0 7 cells for 30 minutes on ice, then washed and suspended in H F N with DNase I. Double-stained cells were first stained with anti-CD 34 and SAM-FITC as above, then washed, incubated with ant i -HLA-DR-PE for 30 minutes at 4°C and finally washed and suspended in H F N with DNase I. Cells were analyzed and sorted on a Becton Dickinson F A C S 440 or a F A C S S t a r l u s using a single argon laser at 488 nm. FITC fluorescence was measured using a 530/30 band pass filter, PI fluorescence using a 630 long pass filter, and P E fluorescence using a 575/26 b a n d pass filter. The F A C S was calibrated prior to each r u n us ing 10 u.m fluorescent microspheres (Coulter Corp. , Hialeah, Florida, U.S .A. ) . Compensation for double-stained specimens was set up in both FITC and PE fluorescence with single-stained samples. Cells were 57 sorted In the full drop envelope mode (FACS 440) or Counter mode (FACS S t a r p l u s ) at 2000 to 3000 cells per second. Data was collected In list mode at 10,000 cells per sample. Cells were maintained at 4°C before during and after the sort and collected In Iscove's 50% FCS medium. Purified LTC-IC for limiting dilution and co-culture experiments (Chapters V and VI) were isolated using the following combination of sorting criteria: low to intermediate FLS and low O L S gates(to include cells with properties similar to small lymphocytes) and high C D 34 expression and very low or negative HLA-DR expression. Clonogenic cells and L T C - I C enrichment relative to unsorted low density cells was calculated by dividing the frequency of the former in the sorted fraction by their frequency in the unsorted, low density suspension. Recovery in a given sorted fraction was calculated by multiplying the calculated enrichment by the percentage of cells i n the fraction sorted. In the experiments where all cells were sorted according to their light scatter properties or fluorescence, the recovery in individual fractions was normalized so that the sum of all fractions i n an individual experiment was 100% to allow comparisons between fractions in different experiments. Differences between the mean recovery of LTC-IC cells in a given fraction and the mean recovery of clonogenic cells in that fraction were compared using a two-tailed Student's t test. (B) Studies using Workshop Antibodies (Chapter IV) Low density cells were first washed in H F N and then 1 0 7 cells per ml were stained with equal volumes of Workshop monoclonal antibodies diluted 400 x in H F N , for 30 minutes on ice. These monoclonal antibodies were obtained as part of our part ic ipat ion i n the F o u r t h International Workshop on Leukocyte Differentiation Antigens. Newly developed monoclonal antibodies are submitted to this Workshop and aliquots sent to participating laboratories for 58 characterization of their reactivity with human leukocytes. Low density bone marrow cells were then washed and stained with SAM-FITC, washed again and suspended in H F N with 2 ug/ml P I and 100 u.g/ml DNase I. Cells were analyzed and sorted within open FLS, and low OLS gates, with exclusion of dead (PI positive) cells. Cells were sorted into a negative and a weakly positive population based on their fluorescence profile as compared to unlabelled stained cells (SAM-FITC only). A third strongly positive fraction was also sorted if s u c h a populat ion was detectable. 3. CLONOGENIC ASSAYS Primit ive erythropoietic ( B F U - E ) , granulopoiet ic ( C F U - G M ) , a n d m u l t i - l i n e a g e (CFU-GEMM) progenitors were determined by assay i n methylcellulose cultures as previously d e s c r i b e d . 9 The plating mixture i n which the cells were cultured consisted of 0.8-0.9% methylcellulose (4000cps) diluted in Iscove's medium supplemented with 30% F C S , 1 m g / m l of deionized bovine serum albumin, 10% v / v agar stimulated h u m a n leucocyte conditioned medium, partially purified erythropoietin (3 U/ml), 1 ml of extra glutamine (2.92gm/100ml) and 1 0 " 4 M 2-mercaptoethanol. Progenitor numbers were derived from counts of colonies made after incubation of the culture for 18 - 21 days at 3 7 ° C . Marrow buffy coat cells were plated at 2 x 10 5 nucleated cells/ml. Marrow cells recovered after Percoll density centrifugation were plated at 5 x 1 0 4 nucleated c e l l s / m l . Sorted fractions were plated at lower cell concentrations depending on the enrichment of progenitors anticipated from preliminary experiments. Al l assays were set up in duplicate or quadruplicate 1.1ml volumes. 4. L O N G - T E R M MARROW CULTURES (LTC) L T C were established and maintained in general according to procedures previously described. 9 Briefly, cells were suspended in LTC medium (alpha medium supplemented with 40 59 m g / l Inositol, 10 m g / l folic acid. 400 m g / l glutamlne, 12.5% horse serum, 12.5% F C S , 1 0 " 4 M 2-mercaptoethanol, and 10" 6 M hydrocortisone sodium succinate) and aliquots were then placed i n 35 m m Corning tissue culture dishes (Corning Glassworks, Corning , N.Y. , U.S .A. ) on pre-established, irradiated, normal marrow feeders. To prepare feeders, allogeneic marrow was used to initiate primary L T C i n 60 m m tissue culture dishes two to six weeks prior to the experiment and these were then maintained as for regular L T C . One to seven days prior to setting up co-cultures, nonadherent cells were removed and the adherent cells t rypsinized, 9 irradiated with 15 Gray (Gy) of 250 KVp X-rays, and plated in 35 m m Corning tissue culture dishes at 3 x 10 5 cells per dish (i.e. 3 x 10 4 cells per c m 2 ) . 1 0 Unprocessed marrow or marrow buffy coat cells were plated at 8 x 1 0 6 cells per 2.5 ml L T C , low density cells at 10 6 cells per 2.5 ml LTC, and sorted fractions at proportionately lower cell concentrations depending on the degree of enrichment anticipated from preliminary experiments. Cultures were incubated for the first 3 to 4 days at 3 7 ° C and thereafter at 33°C , in an atmosphere of 5% CO2 i n air. At weekly intervals, half of the nonadherent cells were removed and at the same time half of the medium was replaced with fresh medium. After 5 weeks all nonadherent cells were removed and the cells in the adherent layer suspended by trypsinization. 9 Nonadherent and adherent cells were then washed and aliquots assayed for clonogenic cell content. 5. LIMITING DILUTION ANALYSIS USING L O N G - T E R M C U L T U R E S Absolute frequencies of LTC-IC were determined using m i n i - L T C and limiting dilution techniques. Mini LTC were established and maintained in general as above but with appropriate scaling down of the number of cells used and the volume of medium added each week according to the surface area of the culture dish or well. Low density cells were seeded at 10 3 to 10 5 cells per c m 2 and sorted cells at lower concentrations (30 to 1 0 3 cells per cm 2 ) depending on the 60 anticipated enrichment of LTC-IC. For limiting dilution experiments, L T C were established in 6 m m wells in 96 well flat bottomed Nunclon microwell plates (Nunc, Roskilde, Denmark) with 100 ul of LTC media per well, and these were then seeded with 500 to 10 4 low density cells per well or 10 to 400 of the sorted cells per well. In these analyses each cell suspension was seeded at 3 or 4 different ini t ial cell concentrations with a m e a n of 23 ± 1 replicate wells per concentration. Except where specified, all L T C were initiated by seeding the test cells on irradiated subcul tured L T C adherent layers as described above. After 5 (or 8 weeks as indicated) the nonadherent cells and the adherent cells suspended by trypsinization were washed, pooled, and all cells plated in standard methylcellulose assays to determine the total clonogenic cell content of each L T C (i.e. the number of B F U - E , C F U - G M , and C F U - G E M M progenitors). The frequency of LTC-IC in the starting population was then determined from the frequency of negative wells (no clonogenic progenitors detectable 5 weeks later) at the various cell dose concentrations. Using Poisson statistics and the weighted mean m e t h o d 1 1 , 1 2 with iterative procedures, the best linear fit and standard errors of this function were determined. The frequency of LTC-IC in the starting population was then calculated as the reciprocal of the concentration of test cells that gave 37% negative cultures. 6. RETROVIRALLY INFECTED M2-10B4 CELLS M2-10B4 cells genetically engineered to produce h u m a n G M - C S F , G - C S F or IL-3 by retrovirus-mediated gene transfer were kindly provided by Dr . D o n n a H o g g e . 4 , 1 3 , 1 4 By standard techniques and hybridization of blots to 3 2 P-oligolabelled G M - C S F , G - C S F or IL-3 cDNA probes, these cells had been shown by Dr. Hogge to contain grossly intact proviral D N A and to produce the expected full length and spliced retroviral t r a n s c r i p t s . 1 5 , 1 6 M2-10B4 feeders were prepared prior to the initiation of co-cultures by seeding 3 x 10 5 M2-10B4 cells into 35 m m Corning tissue culture dishes (Corning Glassworks, Corning N.Y.) or into Nunc 96 well plates at 1 0 4 cells per well . In cul tures containing cells f rom more t h a n one growth 61 factor-producing cell line (to test the effect of specific combinations of growth factors), equal numbers of each of the types of cells were used keeping the total cells plated constant at the values given above. All M2-10B4 feeders were irradiated with 80 Gy of 250 kVp X-rays. 7. C O - C U L T U R E S USING ENGINEERED M2-10B4 CELLS In a total of 16 experiments, 800 to 11,000 sorted human bone marrow cells enriched for LTC-IC were placed in cultures with or without feeders (as indicated) in L T C medium. Cultures were then maintained at 3 3 ° C for 5 weeks with weekly half-medium changes. At the end of 5 weeks, all nonadherent cells were removed and counted, and the adherent cells were then suspended by trypsinization. Aliquots equal to 1/3 to 1/2 of total adherent and nonadherent cells were plated in standard methylcellulose cultures for assessment of erythropoietic (BFU-E), granulopoietic (CFU-GM) and multi-lineage (CFU-GEMM) progenitors. Aliquots equal to 1/2 to 2/3 of the cells were also re-seeded on top of irradiated normal human marrow feeders in 96 well plates for assessment of LTC-IC content by limiting dilution analysis and measurement of total clonogenic cell content after an additional 5 weeks. 8. GROWTH FACTOR BIOACITvTrY Growth factor bioactivity was measured in growth media collected 2 days after a complete change of the medium in confluent cultures of viral producer cells or feeders, and in media removed from co-cultures at weekly intervals. Bioactivity was measured by comparing the stimulation of 3 H-thymidine incorporation into appropriate growth factor-responsive cell lines to that obtained with recombinant growth factor standards. Highly purified recombinant G M - C S F and IL-3 were gifts from Biogen (Genova, Switzerland) and Behring (Frankfurt, W. Germany); the recombinant IL-6 used in these assays was purchased from R & D Systems, Inc. (Minneapolis, MN); recombinant G - C S F was purchased from Amersham (Oakville, Canada). G M - C S F and IL-3 62 levels were measured using human A M L 193 cells, G-CSF using NFS 60 cells and IL-6 using B9 cells. 63 R E F E R E N C E S 1. Frickhofen N, Heit W, Heimpel H : Enrichment of hematopoietic progenitor cells from human bone marrow on Percoll density gradients. Blut 44:101, 1982 2. Roos D , deBoer M : Purification and cryopreservation of phagocytes from h u m a n blood, in Sabato G D , Everse J (eds): Methods in Enzymology, vol 132. Orlando, Academic Press, 1986. pp 225 3. M a n n R, Mulligan R C , Baltimore D: Construction of a retrovirus packaging mutant and its use to produce helper-free defective retrovirus. Cell 33:153, 1983 4. Lemoine F M , Humphries RK, Abraham S D M , Krystal G, Eaves C J : Partial characterization of a novel stromal cell-derived pre-B-cell growth factor active on normal and immortalized pre-B cells. Exp Hematol 16:718, 1988 5. Welte K, Platzer E , L u L, Gabrilove JL , Levi E , Mertelsmann R, Moore MAS: Purification and biochemical characterization of human pluripotent hematopoietic colony-stimulating factor. Proc Natl Acad Sci U S A 82:1526, 1985 6. Weinstein Y, Ihle J N , Lavu S, Reddy EP : Truncation of the c-myb gene by a retroviral integration in an interleukin 3-dependent myeloid leukemia cell line. Proc Natl Acad Sci U S A 83:5010, 1986 7. H u m p h r i e s R K , Eaves A C , Eaves C J : Characterization of a primitive erythropoietic progenitor found in mouse marrow before and after several weeks in culture. Blood 53:746, 1979 8. Lansdorp P M , Aarden LA, Calafat J , Zeiljemaker WP: A growth-factor dependent B-cell hybridoma, in Potter M , Melchers F (eds): Current Topics in Microbiology and Immunology. Berlin, Heidelberg, Springer-Verlag, 1986, pp 105 9. Coulombel L, Eaves A C , Eaves C J : Enzymatic treatment of long-term h u m a n marrow cultures reveals the preferential location of primitive hemopoietic progenitors i n the adherent layer. Blood 62:291, 1983 10. Eaves A C , Cashman J D , Gaboury LA, Kalousek DK, Eaves C J : Unregulated proliferation of primitive chronic myeloid leukemia progenitors in the presence of normal marrow adherent cells. Proc Natl Acad Sci USA 83:5306, 1986 11. Porter E H , Berry R J : The efficient design of transplantable tumour assays. Br J Cancer 17:583, 1963 12. Taswell C : Limiting dilution assays for the determination of immunocompetent cell frequencies. I. Data analysis. J Immunol 126:1614, 1981 13. Hogge D E , Cashman J D , Humphries RK, Eaves C J : Differential and synergistic effects of human granulocyte-macrophage colony stimulating factor and human granulocyte colony stimulating factor on hematopoiesis in human long-term marrow cultures. Blood (in press) 14. Otsuka T, Thacker J D , Cashman J D , Eaves A C , Eaves C J , Hogge D E : Microenvironmental presentation of h u m a n IL-3 is required for the stimulation of very primitive h u m a n progenitors in long-term marrow cultures. Exp Hematol 18:569, 1990 (abstr) 64 15. Sambrook J , Fritsch E F , Maniatis T : Molecular cloning: a laboratory manual . Spring Harbor, NY, Cold Spring Harbor Laboratory, 1989, 16. Feinberg AP, Vogelstein B: A technique for radiolabeling D N A restriction endonuclease fragments to high specific activity. Anal Biochem 132:6, 1983 65 C H A P T E R III CHARACTERIZATION AND PARTIAL PURIFICATION O F H U M A N MARROW CELLS CAPABLE O F INITIATING L O N G - T E R M HEMOPOIESIS IN VITRO 1. INTRODUCTION P u r i f i c a t i o n of the most primitive cells i n h u m a n marrow capable of s u s t a i n e d repopulation of hemopoietic tissues In vivo has been hampered by the inability to assay this function of such cells by more direct procedures. Thus strategies for human hemopoietic stem cell purification have had to rely on available in vitro assays, whose validity as assays for cells with repopulating potential has been based on cross-comparisons with similar assays for murine hemopoietic cells. This is possible because pluripotential repopulating cells in murine marrow can be measured in quantitative transplantation assays and procedures that yield murine marrow cell suspensions that are highly enriched in their content of repopulating cells have been described by several g r o u p s . 1 " 4 Analysis of the in vitro clonogenic potential of these highly purified murine cell populations has suggested that most cells capable of long-term repopulation i n vivo may not be detected as clonogenic cells in standard semi-solid culture s y s t e m s . 1 , 5 Nevertheless, most efforts to date to obtain enriched populations of primitive human hemopoietic progenitors have focussed on the use of in vitro clonogenic assays to define selection criteria for repopulating cells on the assumption that they are either identical cells or would be c o - p u r i f i e d . 6 " 9 Such strategies have made it possible to isolate populations from normal human marrow that show a cloning efficiency in vitro of up to 47%. 6 In this Chapter the results of experiments are described i n which the LTC-IC assay (as described in the Introduction and shown schematically in figure 1) was used to characterize the LTC-IC and these characteristics then used as a basis for their enrichment. The characteristics 66 studied were: F L S , O L S , expression of C D 34, and expression of H L A - D R . A s previously discussed the relative number of LTC-IC in a given cell suspension, was assessed by measuring the total (nonadherent plus adherent) number of clonogenic cells present in L T C 5 weeks after their initiation. To eliminate effects due to variable degrees of purification of supportive cells, all L T C were initiated by plating the test cells on pre-established, irradiated normal marrow adherent l a y e r s . 1 0 Low density cells were isolated using discontinuous Percoll density centrifugation. These cells were then analysed and sorted using a F A C S on the basis of their light scatter or cell surface antigen properties. 2. RESULTS (A) Density Separation Some bone marrow cells were from harvests of normal marrow taken from H L A identical siblings of patients requiring marrow for allogeneic bone marrow transplantion (allogeneic marrow). Other samples were from marrow harvests taken f rom patients themselves (autologous marrow). S u c h patients had a variety of malignancies, had received previous chemotherapy, and were undergoing a bone marrow harvest as part of anticipated further treatment involving autologous bone marrow t r a n s p l a n t a t i o n . After Percol l density centrifugation, recovery of nucleated marrow cells < 1.066 g m / c m 3 and < 1.068 g m / c m 3 was 11 ± 3% and 14 ± 6% respectively. Recovery of clonogenic progenitors in these fractions was 73 ± 25% and 94 ± 29% respectively, and recovery of LTC-IC was 49 ± 32% and 97 ± 8%, respectively. Because of the higher recovery of LTC-IC using 1.068 g m / c m 3 , a switch was made to the use of this density in all subsequent experiments. The average enrichment of clonogenic cells was 6 . 5 ± 2 and 9 ± 4-fold, and of LTC-IC was 3 + 1 and 6 ± 3-fold after density separation at 1.066 and 1.068 g m / c m 3 , respectively. Both clonogenic progenitor content and LTC-IC content were lower in suspensions of marrow from autologous donors as compared to normal allogeneic donors; 67 however, when a "t" test was used to compare progenitor content of allogeneic as compared to autologous marrow, the difference in the LTC-IC content of autologous and allogeneic Percoll separated cells was the only one to reach significance (Table I). T A B L E I. Frequency of Clonogenic Cells and LTC-IC in H u m a n Marrow Before and After Density Centrifugation on 1.066 - 1.068 g m / c m 3 Percoll a . Donor Marrow Buffy Coat Clonogenic Cells LTC-IC Marrow Cells < 1.066 - 1.068 g m / c m 3 Clonogenic Cells LTC-IC Allogeneic Autologous 95 ± 14 (5) 63 ± 23 (3) 12 ± 3 (5) 5 ± 1 (2) 640 ± 87 (11) 439 ± 55 (9) 72 ± 13 (11) 20 ± 5 (9) All values shown are the mean ± S E M (n experiments where each experiment was performed on a different patient's marrow) per initial 10 5 nucleated cells plated in methylcellulose on Day 0 (clonogenic cells) or in LTC, in which case the clonogenic cell content was measured 5 weeks later (LTC-IC). Differences between allogeneic and autologous marrow values are not significant except in the case of the reduced LTC-IC concentration found in low density suspensions of autologous marrow harvests (p=0.03). (B) Light Scatter Properties of Clonogenic and LTC-IC Low density marrow cells from 5 individuals (2 allogeneic donors and 3 autologous donors) were sorted into 4 fract ions according to their F L S and O L S properties (Figure 3A). Morphologica l ly , f ract ion I typically contains pr imari ly small lymphocytes and some normoblasts, fraction II contains larger lymphocytes, fraction III contains monocytes, blasts and some promyelocytes and myelocytes, and fraction IV contains primarily maturing granulocytic cells. In each experiment aliquots of cells from the 4 sorted fractions as well as from the starting low density cell suspension were plated in clonogenic assays and were also used to initiate LTC for determination of LTC-IC content. The progenitor recovery in each fraction was determined 68 and then expressed as a percentage of total progenitor recovery for that experiment. The mean recovery of clonogenic cells for each fraction is compared to the corresponding LTC-IC recovery values in Figure 3B. By comparing the percent recovery of LTC-IC and clonogenic cells in each of the 4 fractions analysed, it can be seen that there are significant differences in fractions I and fractions III (p<0.05) with respect to their light scatter characteristics. Selection of cells with low F L S (fraction I) selectively enriched for L T C - I C , whereas sorting of cells in the blast w i n d o w 1 1 (fraction III) selectively enriched for clonogenic cells. This result was not altered when data from autologous or allogeneic marrow harvests were analyzed separately. 69 3A) 40 80 120 160 200 400 800 1200 Forward Scatter 3B) 100 ra c o w Q. I II I I I IV L T C Initiating Cells I II III IV Clonogenic Cells FIGURE 3. Light scatter properties of clonogenic and LTC-IC; 3A) Bivariate probability contour histogram of low density marrow cells, plotting F L S (channel number) versus OLS (channel number) in a representative experiment. 10% of cells fall in the space between each adjacent pair of contour lines; 3B) The mean progenitor recovery ± S E M expressed as a percent of the total number recovered in each of 5 experiments in which the content of LTC-IC and directly clonogenic cells were compared for each of the four fractions sorted. The percent recovery of clonogenic cells for each fraction was compared to the recovery of LTC-IC for the same fraction in a "t" test. Differences between the recoveries of clonogenic and LTC-IC in fractions I and III are significant (p<0.05). 70 (C) HLA-DR Expression on Clonogenic Cells and LTC-IC Low density marrow cells from 6 individuals, 2 autologous donors and 4 allogeneic donors, were stained with a n t i - H L A - D R - P E and sorted into 4 fractions according to their P E fluorescence intensity (Figure 4A). Only cells which had light scatter properties defined by sort fractions I, II or III in figure 3A were included i n the H L A - D R analysis and sorting. The first 2 H L A - D R - P E sort fractions (I and II) overlap with the unlabelled unsta ined control and correspond to 95% and 5% of cells in the control profile, respectively. Fractions III and IV are HLA-DR-positive. Almost all (-90%) of the clonogenic cells were found in the latter 2 fractions. In contrast, approximately half (55%) of the LTC-IC were found in fractions I and II, and these cells were virtually absent from fraction IV (Figure 4B). The differences between clonogenic cell recovery and LTC-IC recovery are significant (p<0.05) in fractions II and IV. 71 Phycoe r y th r i n (HLA-DR ) 4B) 100 75 SO" o l : 60 o 1 40 °" 20 0 I II I II IV I II I I I IV LTC Initiating Cells Clonogenic Cells FIGURE 4. Expression of HLA-DR on clonogenic and LTC-IC; 4A) The fluorescence profile (log scale) of low density, unstained marrow cells (dotted line) is compared to the same , cell suspensions after labelling with anti -HLA-DR conjugated to PE (solid line) in a representative experiment. Relative number of cells is presented versus the phycoerythrin fluorescence channel number. The fluorescence Intensity remained relatively constant between experiments and the gating of fractions I to IV was maintained constant at the fluorescence channels shown; 4B) Mean progenitor recovery ± SEM expressed as a percent of the total number recovered in each of 6 experiments in which the content of LTC-IC and directly clonogenic cells was compared for each of the four fractions sorted. Differences between the recoveries of LTC-IC and clonogenic cells ln fractions n and IV are significant (p<0.05). 72 (D) Progenitor Enrichment by Sorting for High CD 34 Expression Low density marrow cells were also sorted for high C D 34 expression after staining with anti-CD 34 and SAM-FITC. In a first series of experiments, the cells were concomitantly sorted with open FLS gates and low OLS gates (fractions I, II and III in Figure 3A), and the top 5% of viable (Pl-negative) FITC fluorescent cells collected. Table II shows the results obtained for clonogenic cell and LTC-IC numbers per 10 5 cells assayed, and the corresponding enrichment obtained by comparison to unsorted, low density cells. In experiments 3 and 5, it was possible to compare internally the top 5% of C D 34-positive cells with the top 2% of C D 34-positive cells. The frequency of clonogenic cells and their enrichment was not different in the 2 C D 34-positive fractions; however, their recovery decreased approximately 2-fold when only the top 2% of C D 34-positive cells were sorted. In contrast, LTC-IC doubled i n both frequency and enrichment when the top 2% was compared to the top 5% of C D 34-positive cells and the same high recovery was obtained in both fractions. Using Percoll density separation the overall average enrichment of clonogenic cells was 8 ± 3 fold and of LTC-IC was 4.5 ± 2 fold over the original marrow buffy coat cells. Multiplying these numbers by the average enrichment obtained for clonogenic and LTC-IC after sorting for C D 34 (top 5%) gave an enrichment of 260 ± 1 2 0 fold for both clonogenic and LTC-IC as compared to the original marrow bufly coat suspensions. 73 T A B L E II. Degree of Enrichment of Clonogenic and LTC-IC After Sorting of Low Density Human Marrow for High M y l O Expression. Expt No. Top % Sorted Clonogenic Cells L T C - I C a No. Per 10 5 Cells Enrichment vs. Percoll Recovery No. Per 10 5 Cells Enrichment vs. Percoll Recovery 1 5 11,000 25X 44% 2,800 65X 68% 2 5 14,600 68X 47% 600 68X 65% 3 5 11,100 19X 67% 500 19X 67% 4 5 15,100 3IX 70% 1,160 107X 98% 5 5 18,850 23X 58% 2.145 32X 80% Mean 14,100 33X 57% 1,440 58X 76% 3 2 16,100 28X 38% 1,350 50X 69% 5 2 15,600 19X 21% 4,060 6IX 67% Mean 15,850 24X 30% 2,700 56X 68% a Values refer to the number of clonogenic cells in L T C at 5 weeks per 1 0 ° nucleated cells initially plated in LTC. (E) Partial Purification of Clonogenic Cells and LTC-IC Using the FLS, OLS, H L A - D R and C D 34 results obtained above, appropriate gates were then chosen for use i n 4 parameter sorts to selectively isolate either clonogenic or LTC-IC. To enrich for clonogenic cells, cells were sorted for high H L A - D R expression, with or without the addition of high FLS (Table III). A maximum enrichment of 74-fold over the Percoll separated 74 cells was obtained, or ~600-fold enrichment over the concentration of clonogenic cells in the original marrow buffy coat. Such suspensions had an average cloning efficiency of 30 ± 2% and were relatively depleted of LTC-IC. Table IV shows the results obtained using gates to enrich selectively for LTC-IC. In this case cells were sorted for low H L A - D R expression and high C D 34 expression (top 2%) with low or very low FLS. The concentration of LTC-IC was enriched 170- to 190-fold over that measured in Percoll separated cells, and -800-fold over marrow buffy coat. These fractions were found to contain only 0.4 - 0.8% of the low density nucleated cells and only 2% of the clonogenic cells. Recovery of LTC-IC was 50-59%. 100 of these highly purified cells produced, on average, 8 clonogenic progenitors in 5 week-old L T C , even though only 4 of the initial 100 cells were initially capable of colony formation in methylcellulose. A s shown i n Table V the relative proportions of different types of clonogenic cells detected either initially or after 5 weeks in LTC was not significantly different between unsorted low density cells and the fractions most enriched for primitive cells (second and third row. Table IV). 75 T A B L E III. Selective Enrichment of Clonogenic Cells i n Normal H u m a n Marrow by Multi-parameter Sorting. 3 Cells Total Clonogenic Cells L T C - I C b Tested Nucleated Cells Recovery Per Enrich Recovery Per Enrich Recovery (%) 10 5 -ment (%) 10 5 -ment (%) Cells Cells M y l O + ve (Top 5%), 1.3 ± 0 . 2 17,000 32 ± 5 44 ± 12 940 21 ± 7 32 ± 12 H L A - D R High ± 3 , 0 0 0 ± 3 0 (Fraction III) M y l O + ve (Top 5%), 0.9 ± 0 . 6 30,000 74+ 16 78 ± 58 1,700 30 ± 3 26 ± 1 5 HLA-DR High ± 2 , 0 0 0 ± 1 0 0 (Fraction III) FLS-High (Fraction III) Values shown are the mean ± S E M for 7 (first row) and 2 (second row) experiments by comparison to low density Percoll separated cells. Values refer to number of clonogenic cells in LTC at 5 weeks per I O 5 nucleated cells initially plated in LTC. 76 T A B L E IV. Selective E n r i c h m e n t of L T C - I C i n Normal H u m a n Marrow by M u l t i -parameter Sorting. 3 Cells Tested Total Nucleated Recovery (%) Clonogenic Cells Per 10 5 Cells Enrich -ment Recovery Per (%) 1 0 ° Cells LTC-IC 1 Enrich Recovery -ment (%) M y l O + ve (Top 5%) 0.8 + 0.2 13,000 20 ± 3 17 + 6 5,300 110 ± 30 70 ± 30 HLA-DR Low ± 3 , 0 0 0 ± 1 , 6 0 0 (Fraction II) M y l O + ve (Top 2%) 0.4 ± 0.1 4,500 9 ± 3 2 ± 1 7,100 190 ± 60 59 ± 20 HLA-DR Low ± 1 , 1 0 0 ± 2 , 0 0 0 (Fraction II) FLS-Low (Fraction I & II) M y l O + ve (top 2%) 0.4 ± 0.2 3,500 7 + 2 2 ± 1 8,900 170 ± 6 0 50 ± 23 HLA-DR Low ± 1 , 2 0 0 ± 3 , 7 0 0 (Fraction II) FLS-Very Low (Fraction I) a V a l u e s shown are the mean ± S E M for 7 (first row), 6 (second row) and 4 (third row) experiments by comparison to low density Percoll separated cells. D Values refer to number of clonogenic cells in LTC at 5 weeks per 10 5 nucleated cells initially plated in LTC. 77 T A B L E V. Relative Proportions of Different Types of Clonogenic Cells Detected Before and After 5 Weeks in L T C a (% of Total) Clonogenic Cells LTC-IC B F U - E C F U - G M C F U - G E M M B F U - E C F U - G M C F U - G E M M Unsorted Low Density Cells 36 ± 3 62 ± 4 1.2 ± 0 . 2 9 ± 2 91 ± 2 0.8 + 0.3 LTC-IC Enriched (Row 2 and 3 Table IV). 24 ± 5 75 ± 5 0.4 ± 0.3 9 ± 3 90 ± 4 1.4 + 0.8 Values represent mean ± S E M for 10 experiments. Values refer to clonogenic cells derived from LTC at 5 weeks. 3. DISCUSSION Cells in human marrow capable of initiating long-term hemopoiesis in vitro were enriched ~800 fold by a combination of density centrifugation (~4 fold) and a single step 4 parameter F A C S sort (-200 fold). This is shown i n Figure 5 which summarizes the data presented in Tables I, III, and IV. Quantitation of these LTC-IC was based on the measurement of the total number (adherent and nonadherent) of clonogenic progenitors present after a culture period of 5 weeks, on the assumption that a linear relationship exists between the number of clonogenic progenitors present after 5 weeks and the number of cells in the original suspension tested that are capable of generating clonogenic progeny. This assumption was subsequently validated (see Chapter V). For simplicity i n this Chapter, LTC-IC content is given by the total number of clonogenic progenitors present in 5 week-old L T C assays, recognizing that this number is likely be somewhat greater than the number of original LTC-IC (as the latter would each be anticipated to give rise to more than one clonogenic cell detectable after 5 weeks in LTC). 78 (A) (B) (C) Whole BM 120 -90 -(A 5 ° 6 0 -Q. 30 (-1 CFC LTC Enrichment Recovery (%) My-10**, High FLS High HLA-DR 40 -30 <n s W 20 -o> Q. 10 -X CFC LTC 350x 150x 67 18 My-10*** Very Low FLS Low HLA-DR (A 8 CFC LTC 40x 800x 2 35 FIGURE 5: Enrichment of Clonogenic and LTC-IC as compared to whole bone marrow by density centrifugation and FACS sorting. The frequency of clonogenic cells (CFC) and LTC-IC (LTC) in whole buffy coated bone marrow (A) and in fractions maximally purified for clonogenic cells (B) and for LTC-IC (C) are presented. Enrichment and recovery data for these cells in the two sorted fractions are compared to buffy coat by multiplying the results obtained with density centrifugation by the results of FACS sorting. The separate characterization of LTC-IC and directly clonogenic cells with respect to a number of physical and antigenic properties revealed consistent differences that could be exploited to allow their differential enrichment. Previous reports had shown that the modal density of clonogenic progenitors occurs between 1.063 - 1.064 g m / c m 3 . 1 2 This was confirmed here, where essentially all such cells were found to be lighter than 1.066 g m / c m 3 . Less is known about the density profile of LTC-IC, but our results indicate that substantial numbers of these cells may be slightly more dense than clonogenic progenitors. With respect to light scatter 79 properties, previous studies had also shown that the majority of clonogenic progenitors exhibit a high FLS and a low to intermediate O L S 1 3 - 1 4 and are thus concentrated in an area referred to as the blast w i n d o w . 1 1 , 1 5 Again, the present data confirm these findings but additionally show that L T C - I C have a significantly lower F L S than the majority of clonogenic cells, and are therefore found in the light scatter fractions that contain small-medium sized lymphocytes. Coincident with the publication of these finding, Andrews et al also analysed the light scatter properties of LTC-IC using the total content of nonadherent and adherent clonogenic progenitors in 4 week L T C . They found LTC-IC defined in this way to be present in both the low OLS, low FLS (lymphocyte window) and the low OLS, high FLS (blast window) regions. However, although enrichment of these cells was found to be higher in the latter fraction, the former fraction contained the majority of the L T C - I C . 1 5 Expression of the HLA-DR antigen on primitive hemopoietic cells has been a point of some controversy in the literature. 16-20 Most clonogenic cells express detectable levels of H L A - D R antigens, I 6 ' 1 8 although the "blast colony-forming cell" has been found to lack surface HLA-DR a n t i g e n . 2 0 LTC-IC have been reported to both express^ and l a c k 1 7 , 1 9 H L A - D R antigens. In the present experiments, the H L A - D R fluorescence profile was subdivided into 4 fractions, the first 2 corresponding to no or very low H L A - D R antigen expression and the latter 2 to increasingly higher H L A - D R expression. Separate analysis of each of these fractions confirmed that the majority of clonogenic cells express readily detectable levels of H L A - D R , whereas approximately 50% of LTC-IC showed no or very low HLA-DR expression. It is possible that the increasing expression of HLA-DR on LTC-IC may reflect an increasing state of activation of these cells. Sorting of h u m a n marrow cells with high C D 34 antigen expression allowed both clonogenic and LTC-IC to be significantly enriched in agreement with previous observations by o t h e r s . 2 1 ' 2 2 Recent s tudies have suggested that cells responsible for hemopoietic 80 reconstitution in vivo after lethal irradiation are also retained in the C D 34-positive fraction although quantitative measurements to allow enrichment calculations were not performed. 2 2 In the present study, we have obtained evidence that LTC-IC express higher levels of C D 34 than do the majority of clonogenic cells. This is consistent with the previously reported progressive decline in C D 34 antigen expression with increasing hemopoietic cell differentiation at later stages of myelopoiesis. 2 3 Most previous attempts to obtain enriched populations of primitive human hemopoietic cells have focussed on the isolation of cells that form colonies of differentiated myeloid and erythroid cells in semi-solid media containing soluble growth factors within 2 to 3 weeks. The present studies show that the majority of these cells differ in their light scatter properties, and C D 34 and H L A - D R expression from cells that give rise to clonogenic progenitors in the LTC system. Analogous studies with murine marrow would predict that it might be possible to further separate most human LTC-IC from all residual clonogenic cells detected using standard colony assay condit ions. 5 Similarly it may be possible to obtain suspensions that are more enriched in h u m a n clonogenic cells than those described here (30% - Figure 5) which are completely devoid of LTC-IC. Comparative studies of these populations should be useful for the identification of growth factors that stimulate the most primitive hemopoietic cells in normal human marrow and to characterize molecular events that distinguish their response patterns. 81 R E F E R E N C E S 1. Lord BI, Spooncer E : Isolation of hematopoietic spleen colony forming cells. Lymphokine Res 5:59, 1986 2. Visser J W M , de Vries P: Isolation of spleen-colony forming cells (CFU-s) using wheat germ agglutinin and rhodamine 123 labeling. Blood Cells 14:369, 1988 3. Spangrude G J , Heimfeld S, Weissman IL: Purification and characterization of mouse hematopoietic stem cells. Science 241:58, 1988 4. Szilvassy S J , Lansdorp P M , Humphries RK, Eaves A C , Eaves C J : Isolation in a single step of a highly enriched murine hematopoietic stem cell population with competitive long-term repopulating ability. Blood 74:930, 1989 5. M u l l e r - S i e b u r g C E , T o w n s e n d K, W e i s s m a n IL, R e n n i c k D : Pro l i fe ra t ion a n d differentiation of highly enriched mouse hematopoietic stem cells and progenitor cells in response to defined growth factors. J Exp Med 167:1825, 1988 6. L u L, Walker D , Broxmeyer H E , Hoffman R, H u W, Walker E : Characterization of adult human marrow hematopoietic progenitors highly enriched by two-color cell sorting with M y l O and major histocompatibility class II monoclonal antibodies. J Immunol 139:1823, 1987 7. Strife A , L a m b e k C , W i s n i e w s k i D , G u l a t i S, G a s s o n J C , G o l d e D W , Welte K , Gabrilove J L , Clarkson B: Activities of four purified growth factors on highly enriched human hematopoietic progenitor cells. Blood 69:1508, 1987 8. Mouchiroud G, Berthier R, Newton IA, Chapel A : Monoclonal antibodies against human hemopoietic cells and the separation of progenitor cells from bone marrow. Exp Hematol 13:566, 1985 9. Bodger MP, Hann IM, Maclean RF, Beard M E J : Enrichment of pluripotent hemopoietic progenitor cells from human bone marrow. Blood 64:774, 1984 (abstr) 10. Eaves A C , Cashman J D , Gaboury LA, Kalousek DK, Eaves C J : Unregulated proliferation of primitive chronic myeloid leukemia progenitors i n the presence of normal marrow adherent cells. Proc Natl Acad Sci USA 83:5306, 1986 11. C i v i n CI, Loken MR: Cell surface antigens on h u m a n marrow cells: Dissect ion of hematopoietic development using monoclonal antibodies and multiparameter flow cytometry. Int J Cell Cloning 5:267, 1987 12. Frickhofen N, Heit W, Heimpel H : Enrichment of hematopoietic progenitor cells from human bone marrow on Percoll density gradients. Blut 44:101, 1982 13. Atzpodien J , Gulat i S C , Kwon J H , Wachter M , Fried J , Clarkson B D : H u m a n bone marrow C F U - G M and B F U - E localized by light scatter cell sorting. Exp Cell Biol 55:265, 1987 14. Morstyn G, Nicola NA, Metcalf D: Purification of hemopoietic progenitor cells from human marrow using a fucose-binding lectin and cell sorting. Blood 56:798, 1980 82 15. Andrews R G , Singer JW, Bernstein ID: Precursors of colony-forming cells in humans can be distinguished from colony-forming cells by expression of the CD33 and CD34 antigens and light scatter properties. J Exp Med 169:1721, 1989 16. Fa lkenburg J H F , J a n s e n J , v a n der V a k , Veenhof W F , Blotkamp J , Goselink H M , Parlevliet J , van Rood J J : Polymorphic and monomorphic H L A - D R determinants on human hematopoietic progenitor cells. Blood 63:1125, 1984 17. Moore M A S , Broxmeyer H E , Sheridan A P C , Meyers PA, Jacobsen N, Winchester R J : Continuous h u m a n bone marrow culture: l a antigen characterization of probable pluripotential stem cells. Blood 55:682, 1980 18. Falkenburg J H F , Fibbe WE, Goselink H M , van Rood J J , Jansen J : Human hematopoietic progenitor cells i n long-term cultures express H L A - D R antigens and lack H L A - D Q antigens. J Exp Med 162:1359, 1985 19. Keating A , Powell J , Takahashi M , Singer J W : The generation of h u m a n long-term marrow cultures from marrow depleted of la (HLA-DR) positive cells. Blood 64:1159, 1984 20. Brandt J , Baird N, Lu L, Srour E , Hoffman R: Characterization of a human hematopoietic progenitor cell capable of forming blast cell containing colonies in vitro. J C l i n Invest 82:1017, 1988 21. B a i n e s P, M a y a n i H , B a i n s M , F i s h e r J , Hoy T , J a c o b s A : E n r i c h m e n t of C D 3 4 (My 10)-positive myeloid and erythroid progenitors from human marrow and their growth i n c u l t u r e s s u p p l e m e n t e d with recombinant h u m a n g r a n u l o c y t e - m a c r o p h a g e colony-stimulating factor. Exp Hematol 16:785, 1988 22. B e r e n s o n R J , Andrews R G , Bensinger WI, Kalamasz D , Knitter G , B u c k n e r C D , Bernstein ID: Antigen C D 3 4 + marrow cells engraft lethally irradiated baboons. J Clin Invest 81:951, 1988 23. Strauss L C , Rowley SD, La Russa V F , Sharkis SJ , Stuart RK, Civin CI: Antigenic analysis of hematopoiesis . V . Character izat ion of M y - 1 0 antigen expression by n o r m a l lymphohematopoietic progenitor cells. Exp Hematol 14:878, 1986 83 C H A P T E R I V DIFFERENTIAL EXPRESSION O F ANTIGENS ON C E L L S T H A T INITIATE HEMOPOIESIS IN L O N G - T E R M H U M A N MARROW C U L T U R E 1. INTRODUCTION Investigation and purification of primitive human hemopoietic cells capable of long-term repopulation in transplant recipients has been difficult due to the lack of quantitative in vitro assays to detect these cells. Recent studies have shown that the in-vivo repopulating cells may share characteristics with LTC-IC which differ from the majority of directly clonogenic c e l l s . 1 - 5 To determine whether LTC-IC and clonogenic cells might be distinguished by other parameters, several newly developed antibodies submitted to the "myeloid" or "activation" panels of the Fourth International Workshop on Leukocyte Differentiation Antigens were studied. Fourteen antibodies were selected on the basis of their ability to react with activated T cells and myeloid cell lines but not with resting T cells on the assumption that such antibodies might recognize antigens that would not be present on very primitive, quiescent hemopoietic cells but might appear in association with their entry into a cycling state. Such antigens might therefore be expected to be present on most clonogenic cells but not on LTC-IC in normal human marrow aspirates. 2. RESULTS Of the 14 Workshop monoclonal antibodies tested, 2 showed reactivity with both clonogenic and LTC-IC (Table VI). The weakly positive fraction that included the majority of both of these types of cells contained only 2% of nucleated cells in the case of M89 (My 10) but 84 54% In the case of A60 (8F3.9.3). Three monoclonal antibodies A101 (Nu-TfR2), M51 (VIM3) and A40 (MLR3) were unreactive with both types of cells. The remaining 9 showed some ability to distinguish between clonogenic and LTC-IC, being unreactive with majority of LTC-IC but reactive with the majority of clonogenic cells (Table VI). Subpopulations of cells were sorted as outlined in Figure 6. The most potentially useful antibodies, M86 (DF1516), M84 (DF1513) and M92 (VIP-1) were unreactive with only 0-5% of clonogenic progenitors, whereas 48 - 95% of LTC-IC were recovered i n the initial negative fraction. A23 (138-18), A86 (JMC-H9) and A45 (MEM-75) showed an intermediate ability to discriminate these two types of cells with 14 - 22% of clonogenic progenitors unreactive and 52 - 92% of LTC-IC also unreactive. The least useful antibodies in this group included A70 (BU-55), A28 (120-2A3) and A69 (BU-54) which were unreactive with 27 - 42% of clonogenic progenitors and also unreactive with 66 - 100% of LTC-IC (Figure 6). T A B L E VI. Percentage of Clonogenic and L T C - I C in Fractions Sorted According to Staining with Workshop Monoclonal Antibodies Antibody Name Workshop Code Clonogenic Cells LTC-IC Fluorescence No. of Negative Weakly Expts +ve I II Strongly No. of +ve Expts m&rv Fluorescence Negative Weakly +ve II Strongly +ve III&IV A. Both Reactive M y l O 8F3.9.3 M89 A60 1 2 7 54, 8 93 46, 91 22 10 78 89 B. Clonogenic Cell Reactive, LTC-IC Unreactive DF1516 M86 1 0 3 97 2 2, 93 61. 7 37, 0 DF1513 M84 1 3 92 2 2 94. 93 0 ,7 4 , 0 VIP-1 M92 1 5 89 6 1 58 36 5 138-18 A23 2 20, 7 34, 81 46, 12 3 92, 33, 96 7, 65, 4 1 .2 ,0 JML-H9 A86 2 10, 25 79, 75 11, 0 1 52 48 0 MEM-75 A45 1 22 65 13 1 92 7 1 BU-55 A70 2 4, 49 71, 48 25, 3 1 66 34 0 120-2A3 A28 1 37 61 2 1 100 0 0 BU-54 A69 1 42 52 5 1 91 9 0 C. Both Unreactive Nu-TfR2 VIM3 MLR3 A101 M51 A40 78 96 68 20 4 32 94 100 64 3 0 36 86 FLUORESCENCE INTENSITY F I G U R E 6: F l u o r e s c e n c e pro f i l e s of l i g h t - s c a t t e r ga ted l o w d e n s i t y h u m a n b o n e m a r r o w ce l l s s t a i n e d i n d i r e c t l y w i t h W o r k s h o p m o n o c l o n a l a n t i b o d i e s . T h e prof i l e s p r e s e n t e d are those of a n t i b o d i e s t h a t a p p e a r u s e f u l to d i s t i n g u i s h c l o n o g e n i c c e l l s f r o m L T C - I C . V e r t i c a l b a r s i n d i c a t e the w i n d o w s t h a t were u s e d for s o r t i n g w i t h f r a c t i o n I r e p r e s e n t i n g the negat ive c e l l s , f r a c t i o n II r epresent ing the w e a k l y posi t ive cel ls a n d f r a c t i o n III a n d IV represent ing the s t rongly pos i t ive c e l l s . T h e f l u o r e s c e n c e prof i le of u n s t a i n e d ce l l s ( S A M - F I T C only) i s p r e s e n t e d b y the dotted l i n e . T h e r e s u l t s of f u n c t i o n a l assays of the cel ls i n the sorted f rac t ions are g i v e n i n Table V I . 87 3. DISCUSSION Of the 9 monoclonal antibodies found to distinguish between clonogenic and LTC-IC, 8 were subsequently assigned to C D 71 which is the transferrin receptor. One, D F 1516, could not be classified. Taken together with the results described in Chapter III, these findings indicate considerable antigenic disparity between the surface of h u m a n clonogenic cells and LTC-IC. They also illustrate how different antibodies may show different staining patterns even though they react with the same molecule underscoring the limitation of using single antibodies to define cellular expression of particular gene products. The approach described i n this study indicates the ease with which antibodies directed against such differentially expressed determinants can be identified. Together with novel preparative procedures applicable to large numbers of cells expressing C D 3 4 , 6 such antibodies offer significant promise for the further separation of functional subpopulations of very early hemopoietic cells. Such populations of stem cells, purified on the basis of at least two membrane markers, may prove invaluable for studies of early events in normal and leukemic hemopoiesis and may have clinical applications in autologous marrow transplantation. 88 R E F E R E N C E S 1. Winton E F , Colenda KW: Use of long-term h u m a n marrow cultures to demonstrate progenitor cell precursors in marrow treated with 4-hydroperoxycyclophosphamide. Exp Hematol 15:710, 1987 2. Yeager A M , Kalzer H , Santos GW, Saral R. Colvln O M , Stuart RK, Braine H G , Burke PJ , A m b i n d e r R F , B u r n s W H , Ful ler D J , Davis J M , Karp J E , Stratford M , Rowley S D , Sensenbrenner LL, Vogelsang G B , Wingard JR: Autologous bone marrow transplantation i n patients with acute nonlymphocytic leukemia, using ex vivo marrow treatment with 4-hydroperoxycyclophosphamide. N Engl J Med 315:141, 1986 3. Andrews R G , Takahashi M , Segal G M , Powell J S , Bernstein ID, Singer J W : The L4F3 antigen is expressed by unipotent and multipotent colony-forming cells but not by their precursors. Blood 68:1030, 1986 4. Howell A L , Miller R Keefe K A Mclntyre O R Stockner M . Sullivan R, Hibbert S, Noelle RJ , Ball E D : Distribution of the 124-kd antigen defined by monoclonal antibody AML-1-99 on normal and leukemia myeloid cells. Exp Hematol 16:176, 1988 5. Sutherland H J , Eaves C J , Eaves A C , Dragowska W, Lansdorp P M : Characterization and partial purification of human marrow cells capable of initiating long-term hematopoiesis in vitro. Blood 74:1563. 1989 6. Thomas T E , Sutherland H J , Lansdorp PM: Specific binding and release of cells from beads using cleavable tetrameric antibody complexes. J Immunol Methods 120:221, 1989 89 C H A P T E R V FUNCTIONAL CHARACTERIZATION OF INDIVIDUAL HUMAN HEMOPOIETIC S T E M CELLS C U L T U R E D A T LIMITING DILUTION ON SUPPORTIVE MARROW STROMA 1. INTRODUCTION In L T C initiated with murine marrow, lympho-myeloid reconstituting cells are known to be maintained for at least 4 w e e k s 1 " 4 suggesting the potential suitability of analogous human cultures to support the maintenance and measurement of human hemopoietic stem cells with similar properties. As yet it has not been possible to test this prediction directly, as such studies with human cells are necessarily limited to experiments that can be performed in vitro and conditions that support expression of the lymphopoietic potential of the most primitive hemopoietic cells in human marrow have not been identified. 5 In the previous two Chapters it was shown that the number of clonogenic myeloid progenitors present after 5 weeks defines a population of primitive human LTC-IC that can be readily enriched several hundred- f o l d 6 and, at the same time, can be physically separated from the majority (>95%) of the clonogenic progenitors present in the original marrow sample. These experiments did not, however, provide absolute values for the content of LTC-IC in the suspensions tested, nor did they allow an assessment to be made of the proliferative and differentiative potentialities expressed by individual L T C - I C maintained in the L T C system, i.e. in the presence of semi-confluent, irradiated h u m a n marrow adherent (stromal) cell layers. In the present study, L T C were initiated with limiting numbers of input cells to allow investigation of each of these parameters. Figure 7 illustrates the model of hemopoiesis in LTC used to formulate these experiments. 90 o 0) a. (0 o c a) o E 3 10' 2 10' 10' 10 \ Total Clonogenic Progenitors Number of Clonogenic Progenitors per LTC-IC 8 Weeks in Culture F I G U R E 7. Model of hemopoiesis In L T C . The total number of clonogenic progenitors present in a LTC (represented by the dotted line) was postulated to be the result of the behavior of two separable types of cells. Directly clonogenic progenitors (dashed line) contribute to the majority of the clonogenic progenitors i n these cultures during the initial 4 weeks of the culture. LTC-IC (solid line) are present in lower numbers but proliferate to produce clonogenic progenitors which are present in the cultures at > 4 weeks after initiation. Measurement of the number of L T C - I C at initiation (using limiting dilution analysis) and the total number of clonogenic progenitors in the cultures, allows calculation of the average number of clonogenic progenitors produced per LTC-IC. 2. RESULTS (A) Stromal Feeder Requirement of Human LTC-IC In murine L T C , the maintenance of cells that are clonogenic either in vitro or in vivo has been shown to depend on a mechanism involving direct cell-cell Interactions between primitive hemopoietic cells and ontogenetically unrelated stromal c e l l s . 7 , 8 Together these populations constitute a significant proportion of the adherent layer of L T C . 8 , 9 A n adherent layer 91 composed of a mixture of s imilar cell types also forms i n L T C ini t iated with h u m a n m a r r o w 1 0 , 1 1 and the most primitive hemopoietic cells are also found almost exclusively within this f rac t ion . 1 2 Nevertheless, it has been difficult to investigate the importance of the stromal cell component of L T C initiated with h u m a n marrow because of problems i n obtaining functional test populations sufficiently depleted of stromal cells and their precursors. In seven separate experiments the presumed requirement for a functional stromal feeder was evaluated by comparing the 5 week clonogenic progenitor content of cultures initiated by seeding up to 7,000 cells per dish from a sorted ( M y - 1 0 + + , H L A - D R l o w , F L S l o w ) population of low density human marrow cells (highly enriched for LTC-IC 6 ) into dishes with or without pre-established feeder layers of irradiated adherent marrow cells. In culture dishes without feeders no visible adherent layer formed. This was consistently associated with a marked (although not total) and significant (p<0.01. Student's t-test) reduction in the number of clonogenic progeny detectable i n 5 week old cultures (mean value ± 1 S E M = 1.1 ± 0.5% of values in control cultures with feeders). (B) Clonogenic Progenitor Output is Linearly Related to the Number of Marrow Cells  Assayed The relationship between the number of cells placed into a L T C and the number of clonogenic progenitors present 5 weeks later was next examined. Experiments included LTC both with low density marrow cell suspensions, and various subpopulations of M y l O + + cells which, on a per cell basis, yield at least lOOx more clonogenic progenitors after 5 weeks In culture on supportive feeders. The mean number of clonogenic progenitors per LTC at 5 weeks was determined for each concentration at which cells were initially added, and the results then used to calculate the slope of the log of the input:output values. Two examples are shown diagrammatically in Figure 8. The pooled data for all experiments performed with each type of 92 cell suspension, where at least 3 cell concentrations were assessed in any given experiment, are shown in Table VII. In no case was the mean slope value found to differ significantly from 1.0 (p>0.05, t-distribution) T h u s , the relationship between the n u m b e r of clonogenic progenitors detectable after 5 weeks and the n u m b e r of cells assayed did not differ significantly from a linear relationship over a wide range of input cell concentrations. Moreover, this held true regardless of the presence or absence of a variety of mature cell types that are present in the low density fraction of normal marrow and which were removed by the sorting procedure used to enrich for LTC-IC. 93 O y-Q . V> ~o o o 'E 0) O) o c o o 10' 10 3 _ 10 2 _ 10 -1 -My-10++, HLA-DR l o w FLS l o w , < 1.068 gm/cm3 O ' < 1.068 gm/cm' 10 10' 10' 10' 10; 10' 10 Initial Cells per LTC F I G U R E 8. Input-output relat ionship i n L T C . The n u m b e r of clonogenic cells per L T C at 5 weeks is plotted against the n u m b e r of init ia l cel ls seeded into the L T C for a representat ive experiment with Percoll separated cells (open circles, slope of the log of the va lues = 0.91) and a representative experiment with light density. M y l 0 + + , H L A - D R l o w , F L S l o w cells (closed circles, slope of the log of the va lues = 1.02). 94 T A B L E vn. Linearity of Clonogenic Progenitor Numbers After 5 Weeks in LTC as a Function of the Number of Cells Seeded per LTC Cells No. of Mean Slope Probability Expts. ± S E M Slope =1* Percoll separated 11 0.88 + 0.05 p>0.05 (Unsorted) M Y 1 0 + + 2 0.91 +0.06 p>0.2 M Y 1 0 + + 2 1.00 + 0.12 p>0.9 H L A - D R l o w M Y 1 0 + + 7 1.57 + 0.39 p>0.1 H L A - D R l o w F L S l o w * The mean slope observed was not significantly different from 1.0 as determined by a t-distribution. (C) Quantitation of LTC-IC by Limiting Dilution Analysis Although the number of clonogenic cells present after 5 weeks provides a quantitative and hence useful measure of the LTC-IC frequency in the original population, only relative values are obtained. To obtain an absolute measure of these cells, mini-LTC were established i n 96 well plates containing pre-established irradiated adherent layer cells. For each evaluation at least 3 cell concentrations were used with 20 to 24 replicates per concentration. The frequency of negative wells (no clonogenic progenitors detectable 5 weeks later) was then determined and the frequency of LTC-IC in the starting population calculated using Poisson statistics and the weighted mean method l 3 . 1 4 ^ t h iterative procedures to determine the best linear fit and standard errors of this function (Figure 9). Since the Percoll density separation step gives an ~7-fold enrichment in LTC-IC over buffy coat cell suspensions, the frequency of 95 LTC-IC in unseparated bone marrow could be calculated and was found to be ~1 per 2 x 1 0 4 cells. Based on the experiments described i n Chapter III, a 4 parameter F A C S sorting procedure to select cells expressing a high level of M y l O (CD 34), a low or undetectable level of H L A - D R , and showing low OLS properties was used. This yielded a population in which the frequency of LTC-IC was 1-2% (Table VIII). This represents an overall enrichment in these experiments of 200 to 400-fold (by comparison to normal marrow buffy coat). There was no significant difference between the enrichment of L T C - I C i n the M y l 0 + + , H L A - D R l o w cell fractions with or without gating only the F L S l o w cells (p>0.1, Student's t-test). This did, however, consistently eliminate a proportion of directly clonogenic progenitors although, on average, the frequency of clonogenic cells (4.1%) was three times higher than that of LTC-IC (1.3%) in the M y l O + + , H L A - D R l o w , F L S l o w fraction. Nevertheless, in one experiment (#9) the frequency of LTC-IC (13 per 1000) did exceed the frequency of directly clonogenic cells (7 per 1000). In a few experiments duplicate sets of L T C were used to analyse the frequency of cells capable of producing clonogenic progenitors detectable at 8 as well as 5 weeks. Using the 8 week endpoint, the frequency of LTC-IC was, on average, -2-fold lower than that obtained using the 5 week endpoint (Table VIII). 96 Initial Cells per LTC F I G U R E 9. Limiting dilution analysis. Data is from a representative experiment i n which decreasing numbers of light density. My 1 0 + + H L A - D R * 0 W , F L S * o w cells were seeded onto irradiated marrow feeders and the number of clonogenic cells detectable after 5 weeks then determined. In this experiment the frequency of LTC-IC in the starting cell suspension (i.e. the reciprocal of the concentration pf test cells that gave 37% negative cultures) was 1 per 60 cells or 1.7% of all nucleated cells initially present. 97 T A B L E VIII. Absolute Frequencies of LTC-IC No. of % Recoveries LTC-IC frequency Cells expts. Nucl. cells LTC-IC 5 weeks 8 weeks Percoll separated 5 M y l O + + 3 M y l O + + H L A - D R l o w 3 M y l O + H L A - D R l o w 7 F L S l o w 100 100 3.9 ± 0 . 4 76 ± 1 3 0.7 ± 0 . 2 29 ± 1 2 0.8 ± 0 . 1 55 ± 1 9 0.037 ± 0 . 0 0 0.015 ± 0 . 0 0 5 (n=2) 0.58 ± 0 . 2 1 2.3 ± 0.9 0.5 (n = 1) 1.3 + 0.1 1 . 2 ± 0 . 8 (n = 2) * Mean ± S E M expressed as a percent of values in Percoll separated marrow cell suspensions. ** Per 100 nucleated cells in the population tested (Mean ± SEM). *** In a subset of n experiments, duplicate dishes were evaluated after 8 weeks in LTC. (D) Proliferative Properties of LTC-IC To investigate the proliferative potential of L T C - I C , both the average and the range of clonogenic cell numbers in individual 5 week old LTC initiated by limiting numbers of cells was determined. The average number of clonogenic progenitors present at the 5 week time point was 4 and this value remained the same regardless of the purity of the population initially added (p>0.1, analysis of variance) (Table IX). Moreover, from several experiments where duplicate cultures were set up, it was found that the number of clonogenic progenitors per 98 LTC-IC still averaged 4.2 ± 1 . 0 after 8 weeks, a value not significantly different (p>0.1, analysis of variance) from that obtained for 5 week-old cultures. The range in clonogenic cell output values for individual LTC-IC (assessed after 5 weeks of culture) was then determined by analyzing data for only those cultures where the initial concentration of LTC-IC (as determined by data from the entire experiment) was < 0.31 per culture. At this concentration, the likelihood that all of the clonogenic progenitors detected in any given positive culture had derived from a single L T C - I C is >85%. 1 5 The resultant frequency distribution is shown in Figure 10. These data were then used to derive a theoretical frequency distribution of clonogenic progeny numbers expected from individual LTC-IC by subtracting 15% of a derived 2 LTC-IC distribution from the experimental data (see caption to Figure 10). It can be seen that the final derived distribution for single LTC-IC and the data for < 0.31 LTC-IC per culture are, nevertheless, very similar. From the former, it appears that some LTC-IC are capable of producing up to at least 30 clonogenic progenitors. 99 T A B L E IX. Proliferative Potential of LTC-IC Cells Progenitors per LTC-IC * 5 weeks 8 weeks Percoll separated 4.6 ± 0 . 9 5.7 ± 1 . 3 M Y 1 0 + + 3.7 ± 0.9 M Y 1 0 + + 4.4 ± 1 . 0 3.8 H L A - D R l o w M Y 1 0 + + 4.2 ± 0 . 5 3.1 ± 2 . 0 H L A - D R l o w  F L S l o w * Calculated by mult iplying the frequency of L T C - I C i n each experiment (determined by limiting dilution assays) by the total number of cells plated in all L T C to determine the total number of LTC-IC for that experiment. The total content of clonogenic progenitors i n a l l L T C for a n i n d i v i d u a l experiment was obtained directly from clonogenic progenitor assays. Numbers of experiments from which each mean value (± SEM) was derived are the same as in Table VIII. 100 1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 Number of Colonies per LTC F I G U R E 10. Number of clonogenic progenitors produced by individual L T C - I C . The frequency distribution is presented of the number of clonogenic progenitors detected after 5 weeks in a total of 189 L T C each set up by seeding a limiting number of LTC-IC (< 0.31 per LTC) onto pre-established feeders. Positive L T C (n = 47) are those i n which at least 1 clonogenic cell was detected. Experimental data (solid line) is compared to a theoretical frequency distribution of clonogenic progenitors from single LTC-IC (dotted line) derived from the data as follows: Positive cultures set up with < 0.31 LTC-IC per culture have a < 15% chance of having been seeded with > 1 L T C - I C . 1 5 O n the first iteration, we assumed that all the observed cultures were derived from a single LTC-IC. We then calculated the expected distribution of clonogenic cell production from two L T C - I C per culture by tabulating all pairwise combinations of the single cell distribution (multiplying their observed frequencies and adding their clonogenic cell production). A n adjusted single cell distr ibution was then calculated by subtracting for each class in the observed distribution 15% of the calculated two cell distribution for the class. This adjusted single cell distribution was then used to calculate a new two cell distribution which was then used to calculate a second adjusted single cell distribution as previously. The dotted line represents the adjusted single cell distribution after 10 such iterations. 101 (E) Dlfferentlatrve Properties of LTC-Inltiatlng Cells Data from the same LTC initiated with < 0.31 LTC-IC per culture were also analyzed for the type of clonogenic progenitors present after 5 weeks. In the majority of cultures, these were exclusively of the granulocyte-macrophage lineage (Figure 11). However, i n -20% of positive cultures, some progenitors with erythropoietic or multi-lineage potential were seen (i.e. B F U - E and/or C F U - G E M M were detected). Almost all of these cultures also contained some C F U - G M and this association was not independent (p < 0.001, 2-way test of independence), indicating that at least some L T C - I C have the capacity to generate progeny that differentiate along different lineages. co o c CD O i_ CD Q. CD > "55 o Q L c CD O J _ CD a. Only CFU-GM FIGURE 11. Type of clonogenic progenitors produced by individual LTC-IC. The number of 5 week old LTC plated with < 0.31 LTC-IC per culture that were found to contain only C F U -G M , only B F U - E , or a combination of C F U - G M and B F U - E or C F U - G E M M were compared. (No other combinations were observed). Data is from the same LTC analyzed in Figure 10 for total clonogenic cell content. Using a two-way test of independence, the probabiUty that a C F U - G M and a B F U - E or C F U - G E M M occurred together at the frequency observed by chance alone is p < 0.001. 102 3. DISCUSSION In this Chapter a quantitative assay for a very primitive hemopoietic cell l n h u m a n marrow has been described. This cell is identified on the basis of its ability to give rise to progeny clonogenic cells i n the presence of irradiated stromal feeder layers also of h u m a n marrow origin. The assay is l inear over a wide range of input cell n u m b e r s both for unseparated marrow as well as for highly purified cell suspensions. T h i s validates the applicability of this assay to the measurement of LTC-IC numbers in a variety of experimental and clinical situations, and hence opens up opportunities for investigating both intrinsic (genetic) and extrinsic (microenvironmental) parameters that may influence the regulation of very primitive human hemopoietic cells in vitro and in vivo. A s a first application of this approach we have used limiting dilution analysis techniques to quantitate the concentration of these LTC-IC i n normal h u m a n marrow and i n various purified subpopulations assessed using a 5 week clonogenic cell output endpoint. Their frequency in unseparated marrow is ~1 per 2 x 1 0 4 cells, i.e. ~30-fold less than the frequency of clonogenic cells ( C F U - G M plus B F U - E plus C F U - G E M M ) and comparable to frequencies of cells that generate "blast" cell colonies, although reported values for the latter vary widely depending on assay c o n d i t i o n s . 1 6 , 1 7 The use of an 8 week rather than a 5 week culture period preceding assessment of the number of daughter clonogenic cells produced detects a LTC-IC that is somewhat less frequent in normal human marrow. Interestingly, this latter type of LTC-IC (detected using the 8 week end-point) also appears to be more resistant to 4 - H C 1 8 suggesting that it is more primitive. However, whether it is a truly distinct cell type or represents a subpopulation of the LTC-IC identified using the 5 week end-point, cannot be determined from available data since both are co-purified i n the most enriched populations currently obtainable. 6 103 Assessment of the number and type of clonogenic cells present in cultures seeded with limiting numbers of L T C - I C (i.e. as low as 10 cells per well from the most highly purified populations) has also provided information about their proliferative and differentiative capacities. From analysis of a large number of such cultures, the proliferative capacity of individual LTC-IC was found to vary widely even when maintained under the same conditions and assessed at the same time, with some L T C - I C generating up to 30 clonogenic cells detectable after 5 weeks. A similar variability i n the proliferative potential exhibited by individual pluripotent clonogenic cells during colony formation in semi-solid medium has been documented p r e v i o u s l y 1 9 and it has been suggested that this may reflect the operation of a probabilistic mechanism contributing to the regulation of stem cell decisions to undergo terminal differentiation. 2 0 The average number of clonogenic progenitors per LTC-IC assessed after 5 weeks was found to be 4 and the same average value was also obtained for positive 8 week-old cultures. This proliferative function is clearly dependent on the presence of the cells in the irradiated stromal feeder, since highly purified M y l O + + H L A - D R l o w cells in the low FLS and low to medium OLS (lymphocyte) window fail to produce clonogenic cells in the absence of a feeder (and also do not themselves contain stromal cells or their precursors). Although the majority of clonogenic progenitors produced in the presence of a competent adherent layer appeared to be restricted to the generation of granulocytes or macrophages, ~20% of the LTC-IC could also be shown to generate cells with erythropoietic potential. This may represent the true fraction of L T C - I C that are multipotent or an underestimation. Measurements of the type and number of clonogenic cell output are limited by the same reliance on a single time point to evaluate the progeny produced from any given LTC-IC. In addition, it is not known whether the culture conditions used here to detect LTC-IC are optimal for the generation of clonogenic progeny. Indeed, this seems unlikely given the intermittent 104 pattern of primitive progenitor proliferative activity previously shown to occur i n these cul tures . 2 1 The ability to distinguish and hence separate clonogenic cells from a more primitive population from which they derive should facilitate identification of environmental conditions that may influence the earliest steps in human hemopoietic cell development. For example, altered self-renewal and differentiation of L T C - I C can now be separately assessed and quantitated. Such information should serve as a useful starting point for investigating the molecular bas is of how these processes may be u n c o u p l e d i n var ious hematological malignancies. 2 2 105 R E F E R E N C E S 1. Dexter T M , Spooncer E : Loss of immunoreactlvity In long-term bone marrow culture. Nature 275:135, 1978 2. Schrader JW, Schrader S: In vitro studies on lymphocyte differentiation. I. Long-term in vitro culture of cells giving rise to functional lymphocytes in irradiated mice. J Exp Med 148:823, 1978 3. Dorshkind K, Phillips RA: Characterization of early B lymphocyte precursors present in long-term bone marrow cultures. J Immunol 131:2240, 1983 4. Fraser C, Eaves C J , Szilvassy S, Humphries RK: Use of retroviral marking to demonstrate hemopoietic stem cells with lympho-myeloid repopulating ability i n long-term murine marrow cultures. Blood 74:113a, 1989 (suppl)(abstr) 5. L e B i e n T W : Growing h u m a n B-cell precursors i n vitro: The continuing challenge. Immunol Today 10:296, 1989 6. Sutherland H J , Eaves C J , Eaves A C , Dragowska W, Lansdorp P M : Characterization and partial purification of human marrow cells capable of initiating long-term hematopoiesis in vitro. Blood 74:1563, 1989 7. Bentley SA: Close range celhcell interaction required for stem cell maintenance in continuous bone marrow culture. Exp Hematol 9:308, 1981 8. Dexter T M , Spooncer E , Toksoz D , Lajtha L G : The role of cells and their products in the regulation of i n vitro stem cell proliferation and granulocyte development. J Supramol Struc 13:513, 1980 9. Perkins S, F le ischman RA: Hematopoietic microenvironment. Origin , lineage, and transplantability of the stromal cells in long-term bone marrow cultures from chimeric mice. J Clin Invest 81:1072, 1988 10. Eaves A C , Cashman J D , Gaboury LA, Eaves C J : Clinical significance of long-term cultures of myeloid blood cells. C R C Crit Rev Oncol Hematol 7:125, 1987 11. Simmons PJ , Przepiorka D, Thomas E D , Torok-Storb B: Host origin of marrow stromal cells following allogeneic bone marrow transplantation. Nature 328:429, 1987 12. Coulombel L, Eaves A C , Eaves C J : Enzymatic treatment of long-term h u m a n marrow cultures reveals the preferential location of primitive hemopoietic progenitors in the adherent layer. Blood 62:291, 1983 13. Porter E H , Berry R J : The efficient design of transplantable tumour assays. Br J Cancer 17:583, 1963 14. Taswell C : Limiting dilution assays for the determination of immunocompetent cell frequencies. I. Data analysis. J Immunol 126:1614, 1981 15. Coller H A , Coller BS: Poisson statistical analysis of repetitive subcloning by the limiting dilution technique as a way of assessing hybridoma monoclonality, i n Langone J J , V a n Vunakis H (eds): Methods in Enzymology, vol 121. New York, Academic Press, Inc., 1986, pp 412 106 16. Leary A G , Ogawa M : Blast cell colony assay for umbilical cord blood and adult bone marrow progenitors. Blood 69:953, 1987 17. Gordon MY, Dowding CR, Riley GP, Greaves M F : Characterisation of stroma-dependent blast colony-forming cells in human marrow. J Cell Physiol 130:150, 1987 18. Winton E F , Colenda KW: Use of long-term h u m a n marrow cultures to demonstrate progenitor cell precursors in marrow treated with 4-hydroperoxycyclophosphamide. Exp Hematol 15:710, 1987 19. H u m p h r i e s R K , Eaves A C , Eaves C J : E x p r e s s i o n of stem cell b e h a v i o u r d u r i n g macroscopic burst formation in vitro, in B a u m S J , Ledney G D , van B e k k u m D W (eds): Experimental Hematology Today 1980. New York, Karger, 1980, pp 39 20. W u A M , Siminovitch L, Till J E , McCulloch E A Evidence for a relationship between mouse hemopoietic stem cells and cells forming colonies i n culture. Proc Natl Acad Sci U S A 59:1209, 1968 21. C a s h m a n J , Eaves A C , Eaves C J : Regulated proliferation of primitive hematopoietic progenitor cells in long-term human marrow cultures. Blood 66:1002, 1985 22. M i y a u c h i J , Kelleher C A , Y a n g Y - C , Wong G G , C l a r k S C , M i n d e n M D , M i n k i n S, M c C u l l o c h E A : The effects of three recombinant growth factors, IL-3, G M - C S F , and G - C S F , on the blast cells of acute myeloblastic leukemia maintained i n short-term suspension culture. Blood 70:657, 1987 107 C H A P T E R V I DIFFERENTIAL REGULATION OF SEQUENTIAL STAGES O F H U M A N HEMOPOIESIS IN L O N G - T E R M C U L T U R E OF HIGHLY PURIFIED HEMOPOIETIC S T E M C E L L S MAINTAINED ON GENETICALLY ENGINEERED MURINE STROMAL C E L L S 1. INTRODUCTION The hemopoietic growth factors now designated as G - C S F , G M - C S F and IL-3 were ini t ial ly identified by their ability to support colony formation by granulopoietic and multilineage cells in vitro. 1 Subsequent studies with these recombinant human factors have shown that all can stimulate cells at multiple levels of hemopoiesis i n vitro, and in vivo can increase the output of mature g r a n u l o c y t e s 2 - 4 and circulating clonogenic hemopoietic progenitors. 5 Such findings suggest a physiological role for these factors in humans but have provided little information about normal regulatory mechanisms, particularly those that determine the rate of differentiation and self-renewal of primitive hemopoietic cells within the bone marrow. L T C offer a useful model for investigation of these mechanisms since they appear to reproduce i n vitro many of the features of stromal cell-mediated regulation of hemopoiesis attributed to the microenvironment of the marrow in v i v o . 6 , 7 In this Chapter, studies of the role of G - C S F , G M - C S F and IL-3 i n regulating human hemopoiesis in a reconstructed model of the L T C system are described. G - C S F and G M - C S F were selected because their production can be induced from stromal cells present in standard human marrow L T C , and manipulations that alter this production cause quiescent clonogenic hemopoietic cells in the adherent layer to enter S - p h a s e . 8 , 9 IL-3 is not detectable i n such L T C , 1 0 but is a potent direct stimulator of human multi-lineage hemopoietic progenitors as 108 well as differentiating granulopoietic cells in semi-solid a s s a y s . 1 1 Thus, provision of IL-3 via stromal cells to hemopoietic cells in L T C might also influence their behaviour under these conditions. To minimize the role of other factors in the system, a cell suspension highly enriched in LTC-IC, and devoid of stromal elements or their precursors was used to initiate hemopoiesis in the present s t u d i e s . 1 2 , 1 3 The cells were then seeded onto preformed feeder layers that had been previously derived from a cloned, stromal cell line (M2-10B4) of mouse marrow origin and infected with ecotropic retrovirus capable of the transfer and expression of c D N A s for human G-CSF, G M - C S F or I L - 3 . 1 4 M2-10B4 cells were chosen because at the time these studies were being undertaken, M2-10B4 cells were not known to produce any factor that stimulates human hemopoietic c e l l s . 1 5 Through the use of such genetically engineered growth factor-producing feeders it was hoped to reproduce the way in which regulators may be localized in the adherent layer a n d / o r presented to adjacent hemopoietic cells on the assumption that this might influence the nature and magnitude of their e f f e c t s . 1 6 - 1 7 Al l cultures were maintained for 5 weeks and the contributions of G M - C S F - , G - C S F - and IL-3-producing feeders (alone or i n combination) to L T C - I C maintenance (self-renewal), clonogenic cell production (LTC-IC differentiation), and production of nonadherent cells (terminal cell output from intermediate clonogenic progenitor cell types) were then assessed and compared. 2. RESULTS (A) Growth Factor Production by Engineered M2-10B4 Cells Human growth factor producing M2-10B4 cells were generated by infection of the cells with ecotropic retrovirus capable of the transfer and expression of both the neo r gene which renders eukaryotic cells resistant to the neomycin analogue G418 and the cDNAs for either h u m a n G M - C S F , G - C S F or IL-3. When retrovirally-infected M2-10B4 cells had grown to 109 confluence under G418 selection, samples of their growth medium were tested for growth factor bioactivity (Table X). Bioactivity was detected only from cultures of cells infected with the appropriate virus. The levels measured ranged from ~1 to 20 n g / m l as determined by 3 H -thymidine incorporation into responsive cell lines as compared to recombinant controls. These levels remained stable for at least 2 months in the absence of G418 selection even after the cells were irradiated. Growth factor bioactivity in media removed at the time of media change and 3 days after media change from co-cultures of M2-10B4 cells with purified human marrow cells was also detected only from appropriately infected cells and remained essentially unchanged throughout the period of the experiments (data not shown). IL-6 levels in media conditioned by M2-10B4 cells or i n media removed weekly from co-cultures were also m e a s u r e d a n d consistently f o u n d to be < 0.01 n g / m l . In previous experiments the concentration of purified recombinant growth factor required to stimulate half maximal h e m o p o i e t i c c o l o n y growth f r o m n o n a d h e r e n t m a r r o w cel ls p l a c e d i n shor t t e r m methylcellulose assays has been shown to be 0.1 n g / m l for G M - C S F , 10 ng /ml for G - C S F and 1 n g / m l for I L - 3 , 1 4 suggesting that growth factor production by the retrovirally infected M2-10B4 cells was sufficient to warrant testing these cells as feeders in L T C . T A B L E X. Growth Factor Production by Retrovirally-Infected M2-10B4 Cells Retroviral Vector Bioactivity (ng/ml) in Medium G M - C S F G-CSF IL-3 Uninfected or N2 <0.01 <0.1 <0.1 N2-tkGM-CSF 1.0 <0.1 <0.1 N2-tkG-CSF <0.03 20 <0.1 N2-tkIL-3 <0.01 <0.1 6 110 (B) Capacity of M2-10B4 Cells to Support Human Hemopoiesis Total numbers of nonadherent cells, clonogenic cells and L T C - I C i n 5 week-old co-cultures were measured to assess the ability of control (uninfected a n d / o r N2-infected) M2-10B4 cells to support hemopoiesis at these three levels of hemopoietic cell activity (Table XI). Results obtained in each case were compared to those obtained from cultures containing normal h u m a n marrow feeders (MF) or no feeders (i.e. hemopoietic cells seeded directly onto plastic). Despite the lack of detectable G - C S F , G M - C S F , IL-3 or IL-6 in cultures containing control M2-10B4 cells, significant support for all levels of hemopoiesis was evident by comparison to results for cultures without feeders. For LTC-IC maintenance and production of clonogenic cells, M2-10B4 cells were almost as effective as normal h u m a n M F . However, human M F did offer a significant improvement over M2-10B4 cells when effects on terminal cell numbers (nonadherent cell production) were assessed. T A B L E XI. Content of 5 Week Co-Cul tures on M2-10B4 Feeders as Compared to Human M F and No Feeders (Plastic) M2-10B4 Human M F Plastic Nonadherent cells 13 ± 6 28 ± 6* 1.2 ± 0 . 4 * per cell plated (n exp'ts) (15) (13) (12) Clonogenic cells per 4.8 ± 1 . 0 6.2 ± 1.1 0.10 ± 0.03* 100 cells plated (n exp'ts) (15) (14) (15) LTC-IC per 1000 2.3 ± 0.4 3.4 ± 0.8 0 cells plated (n exp'ts) (12) (10) (4) * U s i n g a paired "t" test a significant difference from M 2 - 1 0 B 4 was observed. I l l (C) Specific Growth Factor Effects on Terminal Hemopoiesis Nonadherent cell numbers in 5 week-old cultures containing growth factor-producing M2-10B4 cells were compared in a paired "t" test to cultures with control M2-10B4 cells (Figure 12) and to cultures with human M F . IL-3 producing M2-10B4 cells alone were not different than control M2-10B4 cells (p = 0.4). However, all other types of growth factor-producing feeders, either alone or in combination, significantly increased nonadherent cell output above that seen with control M2-10B4 feeders (p < 0.005). GM-CSF-producing M2-10B4 cells with or without other growth factor-producing M2-10B4 cells were most effective in this regard. They supported the production of -20 times more nonadherent cells than cultures containing control M2-10B4 cells, and - 4 times more nonadherent cells even than cultures containing human M F (p < 0.005). Although the combination of IL-3 and G - C S F producing M2-10B4 cells was less effective for the promotion of terminal cell amplification than any feeder producing G M - C S F , it was equivalent to human M F in this regard. 112 1-m o 7 CM E o CD C CO O O LL t CD (/> CO CD 40 30 20 o £ 10 4 v ^ c r <y <y <5P <^ I 1 I FIGURE 12: The number of nonadherent (NA) cells in 5 week-old co-cultures. Cultures initiated with equal numbers of sorted (light density, C D 3 4 + + , H L A - D R l o w , F L S l o w ) cells seeded onto M2-10B4 feeders producing human growth factors or human M F is compared to NA cells in 5 week-old co-cultures containing control M2-10B4 cells. A paired "t" test was applied to log transformed data from (n) experimental pairs to identify groups that were significantly different (*, p<0.05) from controls. In this and all subsequent analyses a value that was at the limit of detection of the assay as determined by the number of cells assessed was substituted for all zero values. A total of 16 experiments were performed, however data was not available for all feeder combinations for all experiments due to contamination of cultures in some cases, and to a limitation in the number of cells available i n some cases necessitating abbreviation of the experiment. T h u s while multivariate analysis would be possible i n these experiment sets to determine if there was any significant variability in the results overall, further analysis to determine specific significant differences would require complete data sets and therefore would use statistical means to create data to fill all missing data points. Using this analysis less than half of the data would be experimentally derived and thus this analysis, while possible, did not seem to be the most accurate means to analyse this data. We therefore chose to delineate major trends by comparison of growth factor producing feeder combinations to controls using a paired t test. 113 (D) Specific Growth Factor Effects on Clonogenic Cell Output Production of clonogenic cells was analyzed in the same experiments by comparison of numbers of clonogenic cells in 5 week-old co-cultures to controls using a paired "t" test (Figure 13). G - C S F feeders alone and G - C S F plus IL-3-producing feeders provided more support than control M2-10B4 feeders (p < 0.05) and G - C S F plus IL-3 feeders were twice as supportive as human M F , although this latter difference did not quite reach statistical significance (p = 0.12). In contrast, G M - C S F feeders either alone or in combination with G - C S F or IL-3 feeders resulted in clonogenic cell output values at 5 weeks that were not significantly different from those obtained in cultures with control M2-10B4 cells. These findings suggest that G M - C S F + G-CSF or IL-3 is not useful in enhancing clonogenic cell output from LTC-IC in the L T C system. To distinguish whether the IL-3 plus G - C S F producing feeder combination increases clonogenic cells by increasing the number of L T C - I C recruited to differentiate, or by increasing the proliferative ability displayed by the L T C - I C originally present, a n additional series of experiments were undertaken. In these, sorted normal bone marrow cells were seeded at a limiting dilution onto either IL-3 plus G-CSF-producing M2-10B4 cells or human M F and the frequency and average clonogenic cell output by individual LTC-IC was then determined from a knowledge of the clonogenic content of wells measured 5 weeks later. The results from 5 such experiments suggest a slight, although not statistically significant, advantage for the IL-3 plus G - C S F secreting M2-10B4 cells for both parameters assessed, i.e. the proportion of initially seeded cells detected as LTC-IC was 1.4% and 1.1% and the average number of clonogenic cells produced per LTC-IC detected was 5.5 and 4.6 for the IL-3 + G - C S F feeders and human M F respectively. 114 00 o T CM E o t a> V) co a> >_ u c 3 r 2 -4 <vV .NN v x v* v * o c RS O o tf) CO o o CD 2 -3 L F I G U R E 13. The number of clonogenic cells i n 5 week-old co-cultures. Cultures containing M2-10B4 cells producing h u m a n growth factors or h u m a n M F Is compared to control M2-10B4 cells using a paired "t" test on log transformed values from (n) experiments as for NA cells (see caption to Figure 12). 115 (E) Specific Growth Factor Effects on LTC-IC Maintenance By plating cells at limiting dilution on human M F , the absolute number of LTC-IC in a population can be quantitated. 1 2 This analysis can be performed on cells removed at various time points from a culture to provide a measure of the ability of the conditions prevailing in the cultures to promote the maintenance and/or self-renewal of LTC-IC. Approximately 25% of the number of input LTC-IC were detected after 5 weeks in LTC initiated by seeding sorted marrow onto human M F . (Mean ± S E M LTC-IC per 1000 sorted cells originally plated = 16.7 ± 4.0 on day 0, and = 4.3 ± 1.0 at 5 weeks, i n 6 experiments.) The ability of control and growth factor-producing M2-10B4 cells to maintain LTC-IC was similarly assessed by quantitating the number of LTC-IC remaining after 5 weeks in primary cultures containing various feeders. As shown i n Figure 14, the combination of IL-3 plus G - C S F feeders i n the primary cultures allowed significantly better maintenance of LTC-IC than control M2-10B4 feeders (p < 0.05) and was even somewhat better than human MF. GM-CSF-producing feeders alone, or together with G-CSF-producing feeders, provided significantly less LTC-IC maintenance than human M F (p < 0.05) and any culture that contained GM-CSF-producing feeders appeared worse than control M2-10B4 cells for LTC-IC maintenance (p = 0.14 to 0.18). The other feeder combinations tested provided support of LTC-IC maintenance that did not differ significantly from that obtained with human M F or M2-10B4 cells. 116 F I G U R E 14. The number of LTC-IC as determined by limiting dilution analysis i n 5 week-old co-cultures. Cultures initiated with equal numbers of sorted cells seeded onto h u m a n growth factor producing M2-10B4 cells or h u m a n M F is compared to the L T C - I C content of 5 week-old co-cultures containing control M2-10B4 cells using a paired "t" test on log transformed values from (n) experiments, as for NA cells (see caption to Figure 12). 117 (F) Lack of Any Growth Factor Effect on the Proliferative Potential Displayed by LTC-IC  Present After 5 Weeks in Culture In addition to determining the number of LTC-IC maintained under various co-culture conditions, the proliferative potential of these cells, as indicated by the average number of clonogenic progenitors produced per L T C - I C ( C F U / L T C - I C ) before and after culture was measured by limiting dilution analysis. C F U / L T C - I C was the same for LTC-IC maintained on human M F for 5 weeks as for the LTC-IC i n the original purified marrow sample (4.0 ± 0.7 vs 4.3 ± 0.4). Moreover, despite the fact that the number of L T C - I C maintained i n primary co-cultures with various types of growth-factor producing M2-10B4 cells varied from 2-fold higher to 3-fold lower than the number of LTC-IC maintained in cultures containing human M F , the proliferative potential of the LTC-IC present after 5 weeks was not influenced by the type of feeder used in the primary culture (analysis of variance, p=0.46) (Table XII). 118 T A B L E XII. Number of Clonogenic Progenitors Per LTC-IC Harvested From 5 Week-Old Co-cultures Containing Different Types of Feeder Feeder No. of Clonogenic Progenitors Per LTC-IC Human M F 4.0 ± 0.7 (9) M2-10B4 3.3 ± 0 . 7 (11) M2-10B4+G 5.5 ± 1.0 (7) M2-10B4+GM 6.4 ± 2 . 1 ( 5 ) M2-10B4+IL-3 2.8 ± 0.8 (6) M2-10B4+G+GM 3.7 ± 1.1 (5) M2-10B4+IL-3+G 4.7 ± 1.1 (7) M2-10B4+IL-3+GM 4.3 ± 1.0 (6) M2-10B4+IL-3+G+GM 6.0 ± 2.4 (6) G = G-CSF, G M = G M - C S F , (n) = number of experiments 3. DISCUSSION Analysis of specific growth factor effects on different stages of hemopoiesis requires a system where the factors of interest can be tested individually, or in combination, as desired. In addition, separate assays must be available for the direct quantitation of primitive hemopoietic cells and their more differentiated progeny. In this Chapter the development of an experimental strategy that meets both of these requirements is described. This strategy was then used for evaluating the role of 3 growth factors known to exert with unique and distinct effects on different types of human hemopoietic cells in other systems. The strategy involved establishing co-cultures of primitive human hemopoietic target cells with feeder cells from a murine marrow-derived stromal cell line that was not known to secrete growth factors capable 119 of stimulating human hemopoietic cells . 1 £ > These murine cells had been genetically engineered by retroviral-mediated gene transfer to allow elevated levels of endogenously produced human G - C S F , G M - C S F a n d / o r IL-3 to be sustained i n the co-culture system. Because normal h u m a n marrow aspirate samples contain many cells that can also release both G - C S F and G M - C S F in culture, an attempt was made to minimize the presence of such cells. Accordingly, co-cultures were initiated with a subpopulation of human marrow cells that had been highly enriched in LTC-IC (and some clonogenic cells) and depleted of human marrow stromal cells or their precursors, and of monocytes and T cells. Measurements of the number of L T C - I C , clonogenic cells, and nonadherent cells (representing mature granulocytes and macrophages almost exclusively) present i n the co-cultures 5 weeks later then allowed quantitative compar isons of the effect of each growth factor tested on primitive hemopoietic cell self-renewal, generation of clonogenic progeny, and final amplification and differentiation into mature end cells. A summary of the results obtained is shown schematically in Figure 15. LTC-IC Clonogenic cells NA cells IL-3 + G-CSF 2X increase G-CSF IL-3 + G-CSF 3X increase * GM-CSF 15-25X increase • vs control M2-10B4 WEEK 0 WEEK 5 FIGURE 15. Influence of growth factor producing feeders on three levels of hemopoiesis occur lng over 5 weeks i n L T C . Feeders p r o d u c i n g IL-3 p l u s G - C S F promote L T C - I C maintenance, those producing G - C S F with or without IL-3 promote LTC-IC differentiation into clonogenic cells, and those producing G M - C S F promote terminal cell amplification. 120 The least anticipated finding was that none of the 3 human growth factors evaluated was required for the sustained output of cells at any of the 3 levels of hemopoiesis examined. Particularly at the level of the more primitive LTC-IC and clonogenic cells, M2-10B4 cells alone provided effective support not achieved in their absence. It thus seems likely that these murine stromal cells have an as yet uncharacterized growth supporting ability that, like G - C S F , 1 8 crosses species barriers. It has recently been found that M2-10B4 cells produce the factor that binds to the c-kit gene product (C. Eaves, personal communication), like most stromal cell types thus far e x a m i n e d . 1 9 Since this murine factor stimulates primitive human hemopoietic c e l l s , 2 0 its production by M2-10B4 cells may explain the support these cells provide in L T C . The present findings are also consistent with recent reports suggesting that h u m a n myeloid cells can be maintained in vivo in murine recipients without the provision of h u m a n growth f a c t o r s . 2 1 " 2 3 The ability of G M - C S F to greatly amplify the output of mature (nonadherent) cells in vitro above that obtained with standard human M F extends the results of previous experiments in which soluble G M - C S F was added to standard L T C 2 4 , 2 5 or when it was provided by human M F engineered to constitutively produce G M - C S F . 1 4 The design of the present experiments, which allowed the effects of G M - C S F to be examined in the virtual absence of G - C S F or IL-3, suggest that the terminal amplification of granulopoiesis by G M - C S F does not depend on synergy with either of these 2 other factors. However, the combined production of both IL-3 and G - C S F i n the absence of G M - C S F was able to stimulate terminal granulopoiesis as effectively as human M F and was clearly superior to control M2-10B4 feeders or no feeders at all. When the maintenance and differentiation of very primitive cells was assessed, the pattern of growth factor action was different from that seen on mature cell output. G - C S F plus 121 IL-3 allowed the maintenance of LTC-IC to be significantly enhanced, whereas G M - C S F was not effective. Moreover, when G M - C S F was tested together with G - C S F and IL-3, the G M - C S F appeared to negate the enhancing effect of the latter combination. Whether this is due to an ability of G M - C S F to promote LTC-IC commitment to terminal differentiation in a dominant fashion, or to its inhibition of the proliferation of LTC-IC through direct or indirect mechanisms has yet to be determined. Examples of deterministic mechanisms directing hemopoietic cell behaviour have been r e p o r t e d . 2 6 " 2 8 Additional experiments of the present type but focussing on earlier time points should help to explain the basis of the negative effect of G M - C S F on primitive human hemopoietic cells seen here. It is interesting to note that the effects of G - C S F plus IL-3 on L T C - I C maintenance correlate well with the ability of both of these factors to stimulate primitive (high proliferative potential) clonogenic cells to enter S-phase either when added repeatedly to standard L T C of unsorted marrow or when presented by human M F engineered to produce increased levels of G - C S F or I L - 3 . 1 4 - 2 9 (15,36). Additional evidence favouring the very primitive nature of the cell detected by the L T C - I C assay was provided by the lack of decline i n their proliferative c a p a c i t y af ter 5 w e e k s i n c u l t u r e , e v e n u n d e r c o n d i t i o n s t h a t e n h a n c e d t h e i r self-renewal/maintenance during that period. The ability to analyze the differentiation and the maintenance/self-renewal of LTC-IC independently should facilitate future elucidation of the mechanisms that couple these processes to prevent depletion of the stem cell pool when stem cell differentiation is required. It may also permit the identification of mechanisms that allow these processes to be uncoupled either i n disease states or for therapeutic benefit. For example, a factor or combination of factors that could be manipulated to alternatively promote self-renewal at the expense of differentiation and vice versa could replace the need for large marrow transplants, create new 122 opportunities for gene therapy, and provide the beginnings for the creation in vitro of a variety of blood cell products. 123 R E F E R E N C E S 1. Metcalf D : Hemopoietic Colonies. In vitro cloning of normal and leukemic cells. Berlin Heidelberg, Springer-Verlag. 1977, 227 2. Welte K, B o n i l l a M A , Gil l io A P , Boone T C , Potter G K , Gabri love J L , Moore M A S , O'Reilly R J , Souza L M : Recombinant h u m a n granulocyte colony-stimulating factor. Effects on hematopoiesis in normal and cyclophosphamide-treated primates. J Exp Med 165:941, 1987 3. Donahue R E , Wang EA, Stone DK, Kamen R, Wong G G . Sehgal PK, Nathan D G , Clark SC: Stimulation of haematopoiesis in primates by continuous infusion of recombinant human G M - C S F . Nature 321:872, 1986 4. Mayer P, Valent P, Schmidt G , Liehl E , Bettelheim P: The in vivo effects of recombinant h u m a n i n t e r l e u k i n - 3 : D e m o n s t r a t i o n of b a s o p h i l d i f f e r e n t i a t i o n f a c t o r , h i s t a m i n e - p r o d u c i n g activity, and pr iming of G M - C S F - r e s p o n s i v e progenitors i n nonhuman primates. Blood 74:613, 1989 5. S o c i n s k i M A , C a n n i s t r a S A , E l i a s A , A n t m a n K H , S c h n i p p e r L , G r i f f i n J D : Granulocyte-macrophage colony stimulating factor expands the circulating haemopoietic progenitor cell compartment in man. Lancet 1:1194, 1988 6. Dexter T M , Spooncer E , Toksoz D , Lajtha L G : The role of cells and their products in the regulation of in vitro stem cell proliferation and granulocyte development. J Supramol Struc 13:513. 1980 7. Eaves A C , Cashman J D , Gaboury IA, Eaves C J : Clinical significance of long-term cultures of myeloid blood cells. C R C Crtt Rev Oncol Hematol 7:125, 1987 8. C a s h m a n J , Eaves A C , Eaves C J : Regulated proliferation of primitive hematopoietic progenitor cells in long-term human marrow cultures. Blood 66:1002, 1985 9. Cashman J D , Eaves A C , Raines EW, Ross R, Eaves C J : Mechanisms that regulate the cell cycle status of very primitive hematopoietic cells in long-term human marrow cultures. I. Stimulatory role of a variety of mesenchymal cell activators and inhibitory role of TGF-p\ Blood 75:96, 1990 10. Humphries RK, Kay RJ , Dougherty G J , Gaboury LA, Eaves A C , Eaves C J : Growth factor m R N A i n long-term h u m a n marrow cultures before and after addition of agents that induce cycling of primitive hemopoietic progenitors. Blood 72:121a, 1988 (suppl)(abstr) 11. Kannourakis G , Johnson GR: Proliferative properties of unfractionated, purified, and single ce l l h u m a n progeni tor p o p u l a t i o n s s t i m u l a t e d by r e c o m b i n a n t h u m a n interleukin-3. Blood 75:370, 1990 12. S u t h e r l a n d H J , L a n s d o r p P M , H e n k e l m a n D H , Eaves A C , Eaves C J : F u n c t i o n a l characterization of individual human hematopoietic stem cells cultured at limiting dilution on supportive marrow stromal layers. Proc Natl Acad Sci USA 87:3584, 1990 13. Sutherland H J , Eaves C J , Eaves A C , Dragowska W, Lansdorp P M : Characterization and partial purification of human marrow cells capable of Initiating long-term hematopoiesis in vitro. Blood 74:1563, 1989 124 14. Hogge D E , Cashman J D , Humphries RK, Eaves C J : Differential and synergistic effects of human granulocyte-macrophage colony stimulating factor and human granulocyte colony stimulating factor on hematopoiesis in human long-term marrow cultures. Blood (in press) 15. Lemoine F M , Humphries RK, Abraham S D M . Krystal G, Eaves C J : Partial characterization of a novel stromal cell-derived pre-B-cell growth factor active on normal and immortalized pre-B cells. Exp Hematol 16:718, 1988 16. Gordon MY, Riley GP, Watt S M , Greaves M F : Compartmentalization of a haematopoietic growth factor (GM-CSF) by glycosaminoglycans i n the bone marrow microenvironment. Nature 326:403, 1987 17. Roberts R, Gallagher J , Spooncer E , Allen T D . Bloomfield F, Dexter T M : Heparan sulphate bound growth factors: A mechanism for stromal cell mediated haemopoiesis. Nature 332:376, 1988 18. Nicola NA: Granulocyte colony-stimulating factor and differentiation-induction in myeloid leukemic cells. Int J Cell Cloning 5:1, 1987 19. A n d e r s o n D M , L y m a n S D , Baird A , Wignall J M , E i s e n m a n J , R a u c h C , M a r c h C J , Boswell HS, Gimpel S D , Cosman D, Williams D E : Molecular cloning of mast cell growth factor, a hematopoietin that is active in both membrane bound and soluble forms. Cell 63:235, 1990 20. Zsebo K M , W y p y c h J , McNiece IK, L u H S , S m i t h K A , K a r k a r e S B , S a c h d e v R K , Y u s c h e n k o f f V N , Birkett N C , Wil l iams LR, Satyagal V N , T u n g W, B o s s e l m a n R A , Mendiaz E A , Langley K E : Identification, purification, and biological characterization of hematopoietic stem cell factor from buffalo rat liver-conditioned medium. Cell 63:195, 1990 21. M c C u n e J M , Namikawa R, Kaneshima H , Shultz L D , Lieberman M , Weissman IL: The SCID-hu Mouse: Murine model for the analysis of human hematolymphoid differentiation and function. Science 241:1632, 1988 22. Mosier D E , Gulizia R J , Baird S M , Wilson D B : Transfer of a functional h u m a n immune system to mice with severe combined immunodeficiency. Nature 335:256, 1988 23. Kamel-Reid S, Dick J E : Engraftment of immune-deficient mice with human hematopoietic stem cells. Science 242:1706, 1988 24. Haas R, Ogniben E , Klesel S, Hohaus S, Baumann M , Korbling M , Dorken B, Hunstein W: Enhanced myelopoiesis in long-term cultures of h u m a n bone marrow pretreated with recombinant granulocyte-macrophage colony-stimulating factor. Exp Hematol 17:235, 1989 25. Coutinho L H , Will A , Radford J , Schiro R, Testa N G , Dexter T M : Effects of recombinant human granulocyte colony-stimulating factor (CSF), human granulocyte macrophage-CSF, and gibbon interleukin-3 on hematopoiesis in human long-term bone marrow culture. Blood 75:2118, 1990 26. Metcalf D : Clonal analysis of proliferation and differentiation of paired daughter cells: Action of granulocyte-macrophage colony-stimulating factor on granulocyte-macrophage precursors. Proc Natl Acad Sci USA 77:5327, 1980 125 27. Borzillo G V , A s h m u n RA, Sherr C J : Macrophage lineage switching of murine early pre-B lymphoid cells expressing transduced fms genes. Mol Cell Biol 10:2703, 1990 28. Pierce J H , D i M a r c o E , Cox G W , L o m b a r d ! D , Ruggiero M , Varesio L, W a n g L M , Choudhury G G , Sakaguchi AY, D i Fiore PP, Aaronson SA: Macrophage-colony-stimulating factor (CSF-1) induces proliferation, chemotaxis, and reversible monocytic differentiation in myeloid progenitor cells transfected with the human c-fms/CSF-1 receptor cDNA. Proc Natl Acad Sci U S A 87:5613, 1990 29. Otsuka T, Thacker J D , Cashman J D , Eaves A C , Eaves C J , Hogge D E : Microenvironmental presentation of h u m a n IL-3 is required for the stimulation of very primitive h u m a n progenitors in long-term marrow cultures. Exp Hematol 18:569, 1990 (abstr) 126 C H A P T E R V I I SUMMARY AND F U T U R E DIRECTIONS Hemopoiesis is maintained throughout the life of an individual by the proliferation and differentiation of a small subset of cells most of which are located in the bone marrow. Our concepts about the nature of these cells have arisen largely from studies of hemopoietic reconstitution of lethally irradiated animals following the transplantation of limiting numbers of marrow cells. In the last few years considerable hierarchy even amongst cells with in vivo repopulating ability has become evident, the most primitive of which are detected in long-term competitive repopulating a s s a y s . 1 - 5 Characterization of analogous subpopulations of cells in h u m a n bone marrow has been more difficult because in vivo assays are not possible, and murine data have suggested that such cells might not be detectable in standard in vitro colony a s s a y s . 4 " 7 O n the other hand, the cells responsible for initiating hemopoiesis in L T C in the presence of irradiated feeders, which I have called LTC-IC, appear to be more similar to in vivo repopulating c e l l s . 7 , 8 For the work described in this thesis I chose to define LTC-IC as cells that would give rise to at least one daughter cell detectable 5 weeks later as a clonogenic progenitor in a secondary colony assay culture. The choice of the 5 week endpoint was based on earlier studies 9 which showed that 90% of clonogenic cells in normal human marrow were killed by exposure to 100p.g per m l of 4-HC, whereas clonogenic numbers in LTC initiated with these same cell suspensions were 50% of control values in 4 week-old cultures and the same as control in 8 week-old cultures. More recent studies in our c e n t r e 1 0 and elsewhere 1 1 have suggested that cells capable of rapid in vivo hemopoietic recovery may be maintained in LTC for at least 10 days. Such findings provide support for the view that at least some LTC-IC may represent the most primitive hemopoietic elements in the marrow. 127 A s a first approach to testing this hypothesis a number of physical and antigenic characteristics of LTC-IC were defined and compared with clonogenic cells initially present in normal adult human bone marrow. A Percoll density centrifugation step was used to isolate cells with a density of < 1.068 gm/ml to give an enrichment of both clonogenic cells and LTC-IC of approximately 6-fold. These low density cells were sorted into 4 fractions of different FLS and/or OLS properties and each fraction then assayed for its content of clonogenic cells and LTC-IC to allow calculation of the relative enrichment and recovery of both cell types in each case. Most of the LTC-IC were found to have a significantly lower FLS than clonogenic cells. LTC-IC were similarly found to express little or no H L A - D R , in contrast to most clonogenic cells. When the expression of C D 34 on the surface of L T C - I C and clonogenic cells was compared by sorting the top 2% and the top 5% most fluorescent cells stained with a fluorochrome-labelled anti-CD 34 antibody, most LTC-IC were found to be retained in the top 2%, indicating a very high expression of C D 34 and could thus be further distinguished from the majority of clonogenic cells, which were distributed throughout the top 5%. Using a combination of sort gates for FLS, OLS, H L A - D R expression and CD34 expression to optimize for the selective isolation of LTC-IC, an enrichment of approximately 800-fold over marrow buffy coat was obtained. Conversely, other gates allowed the preferential isolation of clonogenic cells. The ability to differentially purify these two populations lends strong support to the concept that the two assays detect largely non-overlapping populations. A series of 14 additional monoclonal antibodies reactive with h u m a n cells but of unknown specificity were screened to determine whether these might reveal further differences between L T C - I C and clonogenic cells. These antibodies had been submitted to the Fourth International Workshop on Leukocyte Differentiation Antigens in the "myeloid" and "activation" sections and had already been shown to react with myeloid cell lines and activated T cells but not resting T cells. Nine detected an antigen which was more highly expressed on clonogenic 128 cells as compared to L T C - I C . Eight of these were later assigned to C D 71 which is the transferrin receptor; one, DF1516, is as yet unclassified. To further validate the use of the LTC-IC assay endpoint to quantitate Initial LTC-IC, the relationship between the number of cells used to initiate hemopoiesis in L T C and the number of clonogenic progenitors produced and hence detectable In clonogenic assays when the LTC were harvested 5 weeks later, was also examined in L T C containing pre-established feeders to eliminate this as a variable. In each experiment progenitor output was clearly related to the original input, and the slope of the relationship overall for unseparated and 3 purified cell popula t ions was i n al l cases not significantly different f rom 1.0 conf i rming a l inear input/output relationship. In the experiments described above, only a relative measure of L T C - I C numbers was obtained. To measure absolute numbers of L T C - I C i n a population, a limiting dilution technique was used . Low n u m b e r s of L T C - I C i n low density or partial ly purif ied cell suspensions were used to establish replicate LTC on pre-established, irradiated feeders in 96 well plates. Each L T C was assessed after 5 weeks to determine the total number of clonogenic progenitors present i n each well. The frequency of LTC-IC was then calculated using Poisson statistics. The frequency of LTC-IC was found to be 1 per 2 x 10 4 in unseparated normal bone marrow and 1 to 2% in the maximally enriched populations. We have thus developed a functional assay that has allowed definition of a primitive hemopoietic cell which is the precursor of clonogenic cells. Current purification strategies have allowed this population to be purified several hundred-fold to achieve a frequency up to 2%. This population will be useful for studies regarding the regulation of LTC-IC as it is relatively depleted of other potential regulatory cells such as stromal cell precursors, monocytes, and T cells all of which can secrete growth factors or provide other potential regulatory signals. 129 However, definitive studies of LTC-IC regulation await studies using single cells or completely pure populations. While it is now possible to use the doner attachment to the F A C S and sort single cells into 10u.l Terasaki plates, there are still significant limitations with the available LTC-IC purity for direct analyses of individual LTC-IC. The amount of work required to analyse 100 cells for every 1 to 2 LTC-IC is enormous and it is of course impossible to distinguish which of the 100 cells is a L T C - I C except retrospectively. Current ly , purified L T C - I C populations still contain twice as many clonogenic cells as LTC-IC, so a proliferative response induced by a growth factor could be the response of either cell type. Clearly a higher degree of LTC-IC purity is desirable, and of particular importance is the elimination of clonogenic cells as these may also respond in short-term studies of LTC-IC regulation. Towards this end I am currently continuing to collaborate i n the identification of cell markers that allow a distinction to be made between clonogenic cells and L T C - I C . For example, I have recently been involved in studies that have shown that LTC-IC have a lower reactivity with Rhodamine-123 as compared to clonogenic c e l l s , 1 2 and that LTC-IC react with anti -CD 45RO but not anti -CD 45R while the majority of clonogenic cells have the reverse reactivity . 1 3 The ability to combine these additional parameters in a single sorting strategy until very recently has been limited by our F A C S capability (which had only one excition laser) and by the low fluorescence of bone marrow cells labelled with a n t i - C D 34 monoclonal antibodies which had been indirectly coupled to fluorochromes. Both of these difficulties have now been overcome with the acquisition of a second laser for our F A C S t a r P l u s and the development of a new high affinity anti -CD 34 monoclonal antibody, 8G12, which can be directly coupled to fluorochromes. 1 4 It is also now possible to consider other types of sorting strategies to obtain more enriched populations of LTC-IC employing a panel of antibodies to selectively deplete C D 34 positive cells that are not LTC-IC. Antibodies directed against C D 3 3 , 1 5 C D 71, C D 1 5 , 1 6 C D 130 45R, and H L A - D R would be examples of potentially useful reagents. T h i s could then be combined with selection of cells that are strongly positive for C D 34 and show low Rhodamine-123 uptake. Such a sorting strategy should be possible using 3 fluorochromes and be within current F A C S capabilities. It is also possible that a pre-FACS enrichment step might be useful; for example the type of column enrichment or depletion techniques developed in the Terry Fox L a b o r a t o r y . 1 7 , 1 8 Development and/or analysis of additional monoclonal antibodies may also reveal reagents that offer significant enrichment possibilities. It is also possible that additional functional studies of L T C - I C may, paradoxically, reveal situations i n which they become enriched due to selectively enhanced self-renewal. Additionally, a specific functional response identifying a LTC-IC might be used to allow their further enrichment if such a response could be detected by F A C S . For example, LTC-IC might up or down regulate cell surface molecules such as growth factor receptors 1 9 or adhesion m o l e c u l e s 2 0 i n response to a specific growth factor and this change might used in a sorting strategy to obtain LTC-IC at an early stage of their differentiation program. The availability of homogeneous populations of LTC-IC would be of enormous importance as this would make possible analysis of the molecules involved in controlling the earliest stages of hemopoiesis and detection of differences in such mechanisms between normal and malignant stem cells. As a first step toward analysis of the regulation of LTC-IC, I began with an investigation of the role of stromal cells in their control. In both murine and human L T C , hemopoiesis can be mainta ined for several months . In the mouse this has been s h o w n to depend on interactions that take place between primitive hemopoietic cells and the marrow stromal cells that are present in the adherent layer of the cul ture . 2 1 To determine whether stromal cells are also essential in human L T C , the extent of hemopoiesis obtainable with highly purified LTC-IC cultured in the presence or absence of pre-established human marrow feeders for 5 weeks was evaluated. The purification scheme used to enrich for L T C - I C clearly eliminated all cells capable of forming an adherent layer since this did not occur even when as many as 7000 131 purified cells were plated on plastic. Moreover, the total clonogenic progenitor content of the culture after 5 weeks was only 1.1% of the value measured in control cultures with feeders. Using the information derived from the limiting dilution experiments, it was possible to determine both the proliferative and differentiative potential of LTC-IC after 5 weeks in LTC on normal human marrow feeders. The average number of clonogenic progenitors produced per LTC-IC was 4, regardless of the purification of the population analysed. By analysis of L T C plated at very low cell concentrations it appears that individual LTC-IC can produce a wide range of clonogenic progenitors, from 1 to approximately 30. It was also possible to demonstrate the multipotent nature of some L T C - I C as both C F U - G M and B F U - E were sometimes present in wells seeded with a single LTC-IC. The molecular mechanisms by which stromal cells influence hemopoiesis has not been well defined in either murine or human systems, although considerable information exists to indicate the Involvement of certain growth factors. This has derived in part from studies of the cycling control of primitive (high proliferative potential) progenitors which are located in the adherent layer of human LTC and which have been shown to be activated into cell cycle by a media change or by stromal cell activators such as IL-ip and P D G F . 2 2 " 2 4 These stromal cell activators also upregulate G - C S F , G M - C S F , IL-6, and I L - i p m R N A a n d growth factor production by stromal cells suggesting this may be part of the mechanism by which stromal cells stimulate hemopoiesis in L T C . 2 4 " 2 6 To investigate the specific role of G-CSF, G M - C S F on the maintenance and differentiation of LTC-IC, I analysed how these functions were altered when highly purified LTC-IC were co-cultured on cells from a murine bone marrow stromal cell line M2-10B4 which had been engineered by retroviral-mediated gene transfer to produce human G - C S F , G M - C S F . At the same time I also undertook similar studies with human IL-3 producing M2-10B4 cells, since IL-3 is known to stimulate very primitive human hemopoietic cells, even though no evidence of IL-3 production in normal L T C has yet been obtained. In 132 addition this approach made possible a similar analysis of combinations of these particular factors. These studies revealed that in the absence of any feeder, all stages of hemopoiesis were reduced markedly (or to undetectable levels) after 5 weeks in LTC and that the uninfected M2-10B4 cells provided significant support for all levels of hemopoiesis and did not differ s ignif icant ly from h u m a n marrow feeders i n their support for the differentiat ion or maintenance of LTC-IC. G M - C S F producing feeders alone, or in combination with G - C S F or IL-3 producing feeders, markedly amplified the later stages of myelopoiesis whereas G - C S F feeders alone, or with IL-3 feeders, provided significantly more support than uninfected M2-10B4 feeders for LTC-IC differentiation (as measured by clonogenic cell output from the co-cultures at 5 weeks). The combination of IL-3 plus G - C S F producing feeders also provided significantly more support for LTC-IC maintenance than uninfected M2-10B4 cells. Moreover, the proliferative potential of the LTC-IC that were detected in 5 week-old co-cultures did not differ from that of LTC-IC in fresh bone marrow (i.e. ~4 clonogenic cells per LTC-IC) nor was there any significant difference in the proliferative potential of LTC-IC maintained on different types of growth factor-producing feeders. T h i s c o - c u l t u r e strategy t h u s allowed a n a l y s i s of p a r t i c u l a r factors or factor combinations that might promote the differentiation or the maintenance/self-renewal of L T C -IC. The additional use of limiting dilution techniques makes it also possible to distinguish between initial recruitment of more LTC-IC (increased plating efficiency of the LTC-IC assay) and elicitation of a greater proliferative response by individual L T C - I C in vitro. My initial investigations regarding the regulation of LTC-IC suggest that these cells do not require any of the previously identified hemopoietic growth factors as their proliferation and maintenance are well supported by a murine stromal cell line that was not known to produce any of these factors. The mechanism of the support of this murine cell line is still unknown; however the very recent discovery of the c-kit ligand as a fibroblast-derived factor of cross-species reactivity for primitive h u m a n hemopoietic cells suggests this as a strong c a n d i d a t e . 2 7 mRNA for this 133 molecule has been identified in murine L T C , 2 8 and Dr. T. Otsuka in the Terry Fox Laboratory has shown that M2-10B4 cells do produce c-kit l igand. Experiments using c-kit ligand monoclonal antibodies (should they become available) or c-kit anti-sense i n knockout experiments, or using S l / S l versus +/+ feeders in co-culture with purified LTC-IC could help to establish the extent to which the c-kit ligand is necessary for L T C - I C differentiation and maintenance. Studies in the mouse certainly indicate that this factor plays a detectable role, but perhaps not an exclusive o n e . 2 9 The fact that this molecule is expressed as a membrane b o u n d m o l e c u l e 3 0 would also help to explain why contact between stromal cells and hemopoietic cells is important in the regulation of hemopoietic cell a c t i v i t y . 2 2 ' 3 1 , 3 2 A considerable dose dependent hemopoietic response has been demonstrated for I L - 3 . 3 3 It is thus possible that variations in the doses of these growth factors or the way in which they are presented might alter either the differentiation or the maintenance of LTC-IC. Evidence that these two processes can be differentially regulated is scanty; however competitive r e p o p u l a t i n g a b i l i t y d o e s d e c l i n e w i t h r a p i d l y r e p e a t e d s e r i a l b o n e m a r r o w t r a n s p l a n t a t i o n s 2 , 3 4 suggesting the possibility that differentiation may be favored over self-renewal under certain conditions. Additionally, evidence of some differential regulation of differentiation and self-renewal has been obtained in studies of the growth factor responses of of leukemic cells from patients with acute l e u k e m i a . 3 5 The ability to recognize mechanisms whereby self-renewal is promoted rather than differentiation may allow for the development of protocols whereby these cells may be amplified in vitro for potential therapeutic purposes. A s information accumulates regarding the applicability of functional assays to detect murine stem cells, it has become clear that the population of cells with long-term i n vivo repopulating ability is quite heterogeneous, and different functional assays may detect only a very primitive subset of these cells, or may detect a more mature subset of these cells and some of their progeny. Even in the murine system it is not clear the exact extent of the overlap 134 between LTC-IC assays and ln vivo repopulating assays. Due to the difficulty i n doing such studies in humans, this relationship might be better clarified in the murine system, perhaps by using retrovirally marked stem cell populations in both competitive repopulation studies and LTC-IC studies. Eventually thetransplantion of marked cells in humans, will be necessary to determine the extent to which the LTC-IC assay detects human repopulating stem cells. 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